Bi-specific antibodies for enhanced tumor selectivity and inhibition and uses thereof

ABSTRACT

A heterodimeric bispecific immunoglobulin molecule includes a first Fab or scFv fragment which specifically binds to EGFR, and a second Fab or scFv fragment which specifically binds to c-MET, and an antibody hinge region, an antibody CH2 domain and an antibody CH3 domain including a hybrid protein-protein interaction interface domain. Each of the interaction interface domains is formed by an amino acid segment of the CH3 domain of a first member and an amino acid segment of the CH3 domain of a second member. The hybrid protein-protein interface domain of the first chain is interacting with the protein-protein-interface of the second chain by homodimerization of a corresponding amino acid segment of the same member of the immunoglobulin superfamily within interaction domains.

FIELD OF THE INVENTION

The present invention concerns bi-specific antibodies, in particularEGFR x c-MET bi-specific antibodies, for enhanced tumor selectivity andinhibition, their use in the treatment of cancer and methods ofproducing the same.

BACKGROUND OF THE INVENTION

Cancer cells are often characterized by an aberrant expression of cellsurface molecules, such as receptor tyrosine kinases one of which is theepidermal growth factor receptor (EGFR). EGFR is activated upon bindingto the Epidermal Growth Factor (EGF) and other growth factor ligands,such as TGF-α, amphiregulin (AR), epiregulin (EP), betacelluin (BC), orHB-EGF (Normanno et al., Gene 366 (2006) 2-16). Upon ligand-induceddimerization and activation, several downstream signaling pathways aretriggered, including RAS/MAPK, PI3K/Akt and STAT that regulate differentcellular processes, including DNA synthesis and proliferation. EGFRsignaling is commonly found deregulated in cancer through differentmechanisms, including genetic mutations of the receptor. Signalingproperties of mutant forms of EGFR in addition also show an alteredcellular trafficking compared to wild type EGFR, since some of theregulatory proteins that balance the EGFR pathway present alteredexpression in cancer. Mutated EGFR is for example found in non smallcell lung cancer (NSCLC) and 60-80% of colorectal cancers express amutated EGFR.

In the advent of anti-EGFR based cancer therapy it was hypothesized thatEGFR targeted therapy would be most effective in tumors overexpressingthe protein, however studies quickly revealed that the levels of EGFRexpression were not correlated with response to anti-EGFR antibodies,such as cetuximab (Liska Clin Cancer Res 17(3) February 2011). IncreasedEGFR gene copy number, overexpression of EGFR ligand and TP53 mutationswere shown to be associated with response to EGFR inhibitors in CRC(Khambata-Ford et al., J Clin Oncol 2007; 25:3230-7; Moroni et al.,Lancet Oncol 2005; 6:279-86; Oden-Gangloff et al., Br J Cancer 2009;100:1330-5; Tabernero J, J Clin Oncol. 2010 Mar. 1; 28(7):1181-9).

Side effects of current EGFR-targeted therapies targeting EGFRoverexpressing cells suffer from toxicities due to basal expression ofEGFR in tissues other than the tumor. For example, cetuximab which is achimeric human-murine monoclonal antibody against EGFR, often causesskin toxicities, a phenomenon which is also observed in EGFR therapywith gefitinib (J Eur Acad Dermatol Venereol, 2010 April; 24(4):453-9);SpringerPlus 2013, 2:22).

Functionally, receptor tyrosine kinases also often times also showredundancy, which will compensate for the loss of one family member. Oneexample is sustained ERBB3 signaling which is observed in some cases ofEGFR mutant tumors treated with gefitinib (Science Vol. 316, 18 May2007: p. 1039-1043). This functional redundancy can ultimately result inacquired tumor resistance to a therapeutic blockade of one family member(Engelmann et al, Science 316, 1039 (2007)). Acquired tumor resistanceoften results in relapse during a RTK inihibitor monotherapy.

Studies revealed that intrinsic resistance to EGFR-targeted therapy canbe the result of downstream effector molecule activation such as KRASwhich is seen in 35%-40% of CRCs (Knickelbein et al, Genes Dis. 2015March: 2(1):4-12). Multiple studies have now shown that KRAS mutationsin CRC confer resistance to cetuximab because of which it is recommendedto limit cetuximab therapy to patients with wild-type KRAS tumors.However, about 25% of colorectal cancer (CRC) patients that arewild-type for KRAS, BRAF, PIK3CA and PTEN do not respond to treatmentwith EGER inhibitors (J Clin Oncol. 2010 Mar. 1; 28(7)1254-61).Molecular analysis of the patients not responding to treatment byBEAMing revealed an amplification of the MET gene in these patientsfollowing treatment (Bardelli et al. Cancer Discov; 3(6): 668-73).Upregulation of hepatocyte growth factor receptor (HGFR, c-MET)expression and of its ligand HGF appears to be one of the major escaperoutes of tumors during EGFR-targeted monotherapy. This is also oftenaccompanied by amplification of the gene encoding c-MET (Engelmann etal. Science 316, 1039 (2007) Clin Cancer Res 2011; 17:472-482). In vitroexperiments with gefitinib treated HCC827 cells revealed a c-METamplification of 5-10 fold (Engelmann et al. Science 316, 1039 (2007)).

The MET gene encodes the for hepatocyte growth factor receptor (HGFR,c-MET), which is a heterodimeric transmembrane receptor tyrosine kinasecomposed of an extracellular α-chain and a membrane-spanning, β-chainlinked via disulfide bonds and which has a single ligand, HGF, alsoknown as scatter factor. Structurally, c-MET comprises several conservedprotein domains, including sema, PSI (in plexins, semaphorins,integrins), 4 IPT repeats (in immunoglobulins, plexins, transcriptionfactors), TM (transmembrane), JM (juxtamembrane), and TK (tyrosinekinase) domains. Binding of HGF to MET triggers receptor dimerizationand transphosphorylation, leading to conformational changes in MET thatactivate the TK domain. C-MET mediates activation of downstreamsignaling pathways, including phosphoinositide 3-kinase (PI3K)/AKT,Ras-Rac/Rho, mitogen-activated protein kinase, and phospholipase C, thatstimulate morphogenic, proliferative, and antiapoptotic activities aswell as stimulating pathways involved in cell detachment, motility, andinvasiveness.

Consistent with the role of c-MET in cell motility and morphogenesis,metastatic lesions typically exhibit higher expression levels of METthan primary tumors (Cipriani at al. Lung Cancer 2009, 63:169-179).Several approaches have been pursued to inhibit either the ligand HBF orthe receptor to inhibit c-MET signaling. For example, AMG102/Rilotumumabbinds preferentially to the mature biologically active form of HGF,interacting with the amino-terminal portion of the β-chain therebyinhibiting HGF binding. Another monoclonal antiobody (mAb) which wasexplored to inhibit HGF activity is Ficlatuzumab. Ficlatuzumab is ahumanized IgG1 antibody that binds HGF ligand with high affinity andspecificity thereby inhibiting c-MET/HGF biological activities.

Rilotumumab has been tested as monotherapy in patients carryingrecurrent glioblastomas, metastatic renal carcinomas or ovarian cancersand in combination with chemotherapy in prostate cancers or withantiangiogenic agents in advanced solid tumors. Ficlatuzumab was testedboth as monotherapy and in association with EGFR inhibitors in NSCLC(Biologics 2013; 7: 61-68). However, a phase II trial with ficlatuzumabdid not reach its primary endpoint.

Thus, despite the fact that progress has been made in the development ofboth, anti-EGFR and anti-c-MET therapies, either as monotherapy or incombination, there is a continued need for improved anti-EGFR cancertherapies, which overcome the current limitations of anti-EGFR basedtherapies and prevent c-MET-driven tumor resistance.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that bi-specificheterodimeric immunoglobulin molecules which bind to both EGFR and c-METare effective in the treatment of EGFR and c-MET-expressing tumors.

In a first embodiment the present invention provides heterodimericbispecific immunoglobulin molecule which comprises

-   -   (i) a first Fab or scFv fragment which specifically binds to        EGFR, and    -   (ii) a second Fab or scFv fragment which specifically binds to        c-MET, and    -   (iii) an antibody hinge region, an antibody CH2 domain and an        antibody CH3 domain comprising a hybrid protein-protein        interaction interface domain wherein each of said interaction        interface domain is formed by amino acid segments of the CH3        domain of a first member and amino acid segments of the CH3        domain of said second member, wherein said protein-protein        interface domain of the first chain is interacting with the        protein-protein-interface of the second chain by        homodimerization of the corresponding amino acid segments of the        same member of the immunoglobulin superfamily within said        interaction domains.        wherein the first or second engineered immunoglobulin chain has        the polypeptide sequence (“AG-SEED”):        GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPX₁DIAVEVVESNGQPENNYKTTP        SRQEPSQGTT TFAVTSKLTX₂DKSRVVQQGNVFSCSVMHEALHNHYTQKX₃ISL (SEQ ID        NO:1), wherein X₁, X₂ and X₃ may be any amino acid.

In one embodiment, in the heterodimeric bispecific immunoglobulinmolecule of the invention the first member of the immunoglobulin superfamily is IgG and the second member is IgA.

In one embodiment X₁ is K or S, X₂ is V or T, and X₃ is T or S in theheterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above

In one embodiment, the first or second engineered immunoglobulin chainof the heterodimeric bispecific immunoglobulin molecule according to theinvention has the polypeptide sequence (“GA-SEED”);GQPREPQVYTLPPPSEELALNEX1VTLTCLVKGFYPSDIAVEVVLQGSQELPREKYLTVVX2PV X3DSDGSX4FLYSILRVX5AX6DVVKKGDTFSCSVMHEALHNHYTQKSLDR, wherein X₁, X₂, X₃, X₄,X₅, and X₆ may be any amino acid.

According to one embodiment, X₁ is L or Q, X₂ is A or T, X₃ is L V, D orT; X₄ is F, A, D, E, G, H, K, N, P, Q, R, S or T; X₅ is A or T, and X₆is E or D in the inventive heterodimeric bispecific immunoglobulinmolecule.

In one embodiment, the first engineered immunoglobulin chain of theinventive heterodimeric bispecific immunoglobulin molecule comprises thepolypeptide sequence (“AG-SEED”):GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPKDIAVEVVESNGQPENNYKTTPSRQEP SQGTTTFAVTSKLTVDKSRVVQQGNVFSCSVMHEALHNHYTQKTISL and the second engineeredimmunoglobulin chain of the inventive heterodimeric bispecificimmunoglobulin molecule comprises the polypeptide sequence (“GA-SEED”):

GQPREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQELPREKYLTWAPVLDSDG SFFLYSILRVAAEDWKKGDTFSCSVMHEALHN HYTQKSLDR.

According to one embodiment, the fast engineered immunoglobulin chain ofthe inventive heterodimeric bispecific immunoglobulin molecule asdisclosed above has the polypeptide sequence (“AG-SEED”):GQPFEPEVHTLPPSREEMTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPSRLEPS QGTTTFAVTSKLTVDKSRVVQQGNVFSCSVNMHEALHNHYTQKSLSL and the second engineeredimmunoglobulin chain of the inventive heterodimeric bispecificimmunoglobulin molecule as disclosed above has the polypeptide sequence(“GA-SEED”):

GQPREPQVYTLPPPSEELALNNQVTLTCLVKGFYPSDIAVEWESNGQPEPREKYLTWAPVLDSDG SFFLYSILRVDASRWQQGNVFSCSVMHEALHN HYTQKSLSL.

In one embodiment, the first Fab or scFv fragment of the inventiveheterodimeric bispecific immunoglobulin molecule as disclosed abovebinds EGFR with an K_(O) of at least 5×10⁻⁸ M.

In one embodiment, the second Fab or scFv fragment of the inventiveheterodimeric bispecific immunoglobulin molecule as disclosed abovebinds c-MET with an K_(D) of at least 5×10⁻⁸ M.

According to one embodiment, the first Fab or scFv fragment of theinventive heterodimeric bispecific immunoglobulin molecule is derivedfrom cetuximab (C225).

In a preferred embodiment, the first Fab or scFv fragment comprises VLand VH sequences selected from the group consisting of SEQ ID NO: 9, SEQID NO:10, SEO ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 43, SEQ ID NO: 44,SEQ ID NO: 45, SEQ ID NO: 46.

In a preferred embodiment, the wherein the second Fab or scFv fragmentcomprises VL sequences selected form the group consisting of SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51

In a preferred embodiment, the VL sequences of the first Fab or scFvfragment of the inventive heterodimeric bispecific immunoglobulinmolecule are selected the VH sequences of said second Fab fragment areselected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 48, SEQ ID NO:50, or SEQ ID NO: 52.

According to a more preferred embodiment, the first and second Fab orscFv fragments of the inventive heterdimeric bispecific immunoglobulinmolecule as disclosed above comprise the amino acid sequences SEQ ID NO:9, SEQ ID NO: 43, SEQ ID NO: 17, SEQ ID NO:18, or SEQ ID NO: 47, SEQ IDNO: 48, or SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 31, SEQ ID NO: 51,SEQ ID NO:32, or SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO:49, SEQ ID NO: 30, SEQ ID NO: 50, or SEQ ID NO: 45, SEQ ID NO:46 SEQ IDNO: 33, SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO: 11, SEQ ID NO: 44,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52.

According to a more preferred embodiment the first and second Fab orscFv fragments of the inventive heterdimeric bispecific immunoglobulinmolecule as disclosed above comprise the amino acid sequences SEQ ID NO:11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, or SEQID NO:45, SEQ ID NO. 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30,SEQ ID NO: 50.

In one embodiment, the Fc domain of the heterodimeric bispecificimmunoglobulin molecule according to the invention interacts with FcRn.

In one embodiment, the amino acids of the inventive heterodimericbispecific immunoglobulin molecule which interact with FcRn are derivedfrom human IgG1.

In one embodiment the inventive heterodimeric bispecific immunoglobulinmolecule as disclosed above mediates antibody-dependent cellularcytotoxicity.

In one embodiment, the invention provides an isolated polynucleotideencoding any of the amino acid sequences as disclosed above.

In one embodiment, the invention provides a vector, which comprises atleast one inventive polynucleotide.

According to one embodiment, the invention provides for a host cellwhich comprises at least one polynucleotide according to the invention,or which comprises at least one vector according to the invention.

In one embodiment, the invention provides a method for producing aheterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above, with the inventive process comprising:

culturing a host cell according to the invention under conditionssufficient for the heterologous expression of said heterodimericbispecific immunoglobulin molecule

purifying said heterodimeric bispecific immunoglobulin molecule

In one embodiment the invention provides the heterodimeric bispecificimmunoglobulin molecule of the invention which is obtainable by theinventive, method as disclosed above.

According to one embodiment, the heterodimeric bispecific immunoglobulinmolecule according to the invention as disclosed above is covalentlycoupled to at least one linker.

In one embodiment the linker of the inventive heterodimeric bispecificimmunoglobulin molecule is coupled to a dye, radioisotope or cytotoxin.

In one embodiment, at least one of the Fab or scFv light chains of theinventive heterodimeric bispecific immunoglobulin molecule is coupled toa dye, radioisotope, or cytotoxin.

In one embodiment at least one linker as disclosed above is covalentlycoupled to at least one of the Fab or scFv light chains of the inventiveheterodimeric bispecific immunoglobulin molecule as disclosed above.

According to one embodiment the inventive heterodimeric bispecificimmunoglobulin molecule comprises two linkers covalently coupled to theFab Of scFv light chains the heterodimeric bispecific immunoglobulinmolecule.

In one embodiment, the Fab or scFv light chains and/or the CH3 domainsand/or the CH2 domains of the inventive heterodimeric bispecificimmunoglobulin molecule are coupled to a linker, whereby said linker iscovalently coupled to a dye, radioisotope, or cytotoxin.

According to one embodiment, the heterodimeric bispecific immunoglobulinmolecule of the invention is for use in the treatment of cancer.

In one embodiment, the inventive heterodimeric bispecific immunoglobulinmolecule is for use in the treatment of cancer.

In one embodiment, the invention provides a composition, which comprisesthe heterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above and at least one further ingredient.

In one embodiment, the invention provides a pharmaceutical compositionwhich comprises the inventive heterodimeric bispecific immunoglobulinmolecule above and at least one further ingredient, or the inventivecomposition as disclosed above.

In one embodiment, the pharmaceutical composition of the invention isfor use in the treatment of cancer.

In one embodiment, the invention provides a method of treating a subjectin need thereof inflicted with cancer, wherein the treatment comprisesadministering to said subject a therapeutically effective amount of theinventive pharmaceutical composition as disclosed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Depicted is the cellular binding on NCI-H441 cells of twoheterodimeric bispecific immunoglobulin molecules of the invention(B10v5x225-H; CS06x225-H) and “one-armed” (oa) heterodimericimmunoglobulin molecules. Anti-HEL anti-hen egg lysozyme (isotypecontrol)

FIG. 2: (A) Epitope binning results, (B) Biosensor experiments usingbio-layer interferometry (cf. Example 3)

FIG. 3: HGF displacement results

FIG. 4: ADCC experiments on A431 cells using the antibodies asindicated.

FIG. 5: Octet analysis of one-armed heterodimeric immunoglobulinmolecule variants (either Fab or scFv). “225-L”, “225-H”, “225-H” denotekinetic variants of humanized cetuximab (hu225), “425” denotes Matuzumab

FIG. 6: Inhibition of c-MET phosphorylation in (A) NCI-H596 cells, (B)in A549 cells.

FIG. 7: Quantitative summary of the c-MET phosphorylation inhibition (A)NCI-H596 cells, (B) A549 cells.

FIG. 8: (A) Inhibition of c-MET phosphorylation in MKN-45 cells usingthe immunoglobulin molecules indicated, (B) inhibition of EGFRphosphorylation in NCI-H596 cells using the immunoglobulin molecules asindicated.

FIG. 9: Cytotoxicity assays on A549 cells. (A) control with no toxinconjugated, (B) assay using Fab-MMAE-CL coupled antibodies as indicated,MMAE: monomethyl auristatin E

FIG. 10: Cytotoxicity assays on (A) EBC-1 cells,. (B) NCI-H441 cells

FIG. 11: Cytotoxicity assay MKN-45 cells which express high levels ofc-Met and moderate levels of EGFR.

FIG. 12: Depicted is the enhanced inhibition of c-MET phosphorylationare HGF-dependent cancer cell lines: (A) NCI-H596, (B) KP-4.

FIG. 13: Enhanced degradation of c-MET following overnight treatmentwith the inventive B10v5x225-H molecule.

FIG. 14: Internalization assay on NCI-H441 cells using the antibodiesand controls as indicated to assess the suitability of individualconstructs for their use as ADC.

FIG. 15: Depicted are the results of a cellular binding assay using theantibody and immunoglobulin molecules indicated.

FIG. 16: Experimental and calculated binding affinity forcomputationally designed point mutants of C225. Letters in superscriptdenote the following: a-The KD (nM) for wild type (C225) and mutant mAbswas determined by surface plasmon resonance (SPR). Where n>1, thestandard deviation is given. Mutations that improved affinity (p<0.01)are in boldface. b-Experimental binding affinity relative to wild type(kcal/mol). c-Predicted binding affinity relative to wild type usingRosetta. d-Predicted change in Rosetta pair energy across the interface.e-Predicted change hydrogen bond energy across the interface.f-Calculated hydrogen bond energy of mutated residue side chain.g-Predicted change in folding energy of the isolated antibody. NQ: NotQuantifiable, very weak binding.

FIG. 17: Kinetic parameters of monovalent parental SEED antibodies incomparison to-MET×EGFR bsAbs binding to soluble c-MET and EGERextracellular domains. Kinetic constants were determined for cetuximaband matuzumab as references. Antibodies were captured by anti-human FcOctet biosensors and binding kinetics were analyzed at indicated analyteconcentrations (25 to 0.8 nM or alternative, 50 to 3.1 nM). Meltingtemperatures (Tm) were determined by thermal shift assays. Legend:n.d.=not determined; KD=affinity constant, ka=association constant;kd=dissociation constant: Tm=melting temperature; oa=one-armed.

FIG. 18: Cell surface receptor densities of human c-MET and EGFR onseveral tumor cell lines from various indications. Keratinocytes(NHEK.f-c.) were used to evaluate EGFR-related skin toxicity and theliver cell line HepG2 for c-MET mediated liver toxicity. Density valuesare presented as mean molecules per cell of triplicates with standarddeviations given in percent. Legend: ACA=adenocarcinoma, CA=carcinoma.

FIG. 19: Inhibition of c-MET and EGFR phosphorylation by c-Met×EGFRbsAbs. IC50 values were calculated upon 3PL fitting of dose-responsecurves using GraphPad Prism. Standard deviations (s.d.) were calculatedfor at least two independent experiments carried out in duplicates.n=number of independent experiments.

FIG. 20: Inhibition of c-MET and EGFR phosphorylation by c-MET×EGFRbsAbs during ligand stimulation. Phosphorylated c-MET (A) andphosphorylated EGFR (B) were quantified in A549, A431 and primarykeratinocytes (NHEK) using electrochemiluminescence assay (ECL). Cellswere treated with varying concentrations of bsAbs and a non-relatedisotype SEED control with subsequent stimulation with 100 ng/ml HGF (A)or 100 ng/ml EGF (B). Triangles indicate respective receptorphosphorylation levels for stimulated (upwards triangle) andnon-stimulated cells (downwards triangle). Dose response curves werefitted using a 3PL model in GraphPad Prism 5 (GraphPad Software, Inc).

FIG. 21: In vitro selectivity of c-MET×EGFR bsAbs in comparison tocetuximab. (A) EBC-1 as tumor model cell line with high to moderatec-MET and EGFR expression and T47D as epithelial model cell line withlow EGFR expression and no c-MET expression were mixed in a ratio of1:30. to order to distinguish the two cell lines, EBC-1 cells werestained with the green membrane dye PKH2. The cell mixture was incubatedwith 300 nM of bsAb and cetuximab and subjected to flow cytometricanalysis. Antibody binding was detected by FITC-labeled anti-hu Fcsecondary antibody. Representative dot plots for green vs. yellowfluorescence are shown. (B) In vitro selectivity was defined as theratio of mean fluorescence intensity of the EBC-1 and the T47D cellpopulation.

FIG. 22: Cytotoxicity of c-MET×EGFR bispecific SEED antibody-drugconjugates generated by covalent, site-directed conjugation of thetubulin inhibitor MMAE C-terminally to both heavy chains in comparisonto cetuximab as ADC and anti-hen egg lysozyme (HEL) ADC as correspondingreference constructs. Cytotoxicity was assessed on EGFR overexpressingtumor cells A431 (A) and MDA-MB-468 (B), on primary keratinocytes(NHEK.f-c., C) as normal epithelial cell line, on c-MET overexpressingcells MKN45 (D) and EBC-1 (E) as well as HepG2 (F) as liver cell line.Assay was run in duplicates in three independent experiments and curveswere fitted by sigmoidal curve fitting using GraphPad Prism 5 (GraphPadSoftware, Inc).

FIG. 23: Cytotoxicity of bispecific c-MET×EGFR ADC on tumor cell lineA431 and keratinocytes. EC₅₀ values for A431 cells and IC₅₀ values forkeratinocytes (NHEK.f-c.) were calculated by sigmoidal curve fittingusing GraphPad Prism 5 (GraphPad Software, Inc). Asterisks indicate poorfitting results because curves do not reach a saturating plateau at thehighest concentration (*). ED₈₀ represents the ADC concentration atwhich 80% of cells are killed in A431 cells in comparison to untreatedcells, TD₂₀ indicates the dose at which cell viability in keratinocytesis reduced by 20%. Two definitions for an in vitro translationaltherapeutic index or therapeutic window were calculated. The differenceof IC₅₀ and EC₅₀ as well as the ratio of TD₂₀ to ED₈₀.

FIG. 24: Analytical SE-HPLC indicates a purity >95% of four exemplarybispecific antibodies (bsAb) following purification: (A) B10v5x225-M,(B) B10v5x225-H, (C) CS06x225-M and (D) CS06x225-H.

FIG. 25: Synergistic effect of CS06x225-H on inhibition of c-MET, EGFRand AKT phosphorylation. (A) A549 cells were incubated with 300 nM ofthe respective mAbs as indicated for 3 h and stimulated with HGF andEGF. Cell lysates were subjected to Western blotting and bothphosphorylated and total EGFR, c-MET, and AKT were detected. GAPDH wasused as a loading control. (B) Quantification of phospho-AKT levels inA549 cells after treatment with 500 nM mAbs as well as combinations ofcontrol mAbs (500 nM each) and stimulation with HGF and EGF. Celllysates were subjected electrochemiluminescence (ECL) ELISA. (C) ECLELISA Of mAbs treated and HGF-stimulated A549 cell lysates forphosphorylated c-MET indicated increased potency of CS06x225-H incomparison to the combination of oa CS06 and oa 225-H. (D) A549 cellswere treated with varying concentrations of mAbs without stimulation andlysates were subjected to ECL ELISA detecting phosphorylated c-METlevels. B10v5x225-M and B10v5x225-H demonstrated comparable partialagonism to LY2875358.

FIG. 26: Internalization of bispecific antibodies (bsAbs) as determinedby flow cytometry and confocal fluorescence microscopy. (A)Internalization was quantified by flow cytometric analysis employing 100nM bsAbs which were detected with anti-human Fc-AlexaFluor488 conjugateat 37° C. for 1 h in comparison to cells incubated at 4° C. Residualcell surface binding was quenched by anti-AlexaFluor488 antibody. (B)EBC-1 cells were incubated with 100 nM CS06x225-H and detected withanti-human Fc-AlexaFluor488 conjugate at 37° C. or 4° C. Surfacestaining was removed by acidic wash.

FIG. 27: Cytotoxicity of bispecific ADCs and bsAb an NHEK after 6 days.Primary keratinocytes (NHEK) were incubated with varying concentrationsof bispecific ADC or alternatively with bsAbs for 6 days, in order toexclude that the slow division rate of keratinocytes in comparison totumor cells influenced cytotoxicity of the tubulin inhibitor MMAE.Curves were blotted using 3PL fitting in GraphPad Prism 5 (GraphPadSoftware, Inc.).

SEQUENCE LISTING

SEQ ID NO: 1 AG-SEED

SEQ ID NO: 2 AG-SEED

SEQ ID NO: 3 GA-SEED

SEQ ID NO: 4 GA-SEED

SEQ ID NO: 5 AG -SEED

SEQ ID NO: 6 GA-SEED

SEQ ID NO: 7 AG-SEED

SEQ ID NO: 8 GA-SEED

SEQ ID NO: 9 humanized C225 V_(L) sequence

SEQ ID NO: 10 humanized C225 VL kinetic variants

SEQ ID NO: 11 humanized C225 VH sequence

SEQ ID NO: 12 humanized C225 VH kinetic variants

SEQ ID NO: 13 humanized C425 VL sequence

SEQ ID NO: 14 humanized C425 VH sequence

SEQ ID NO: 15 c-MET binder A12 VL sequence

SEQ ID NO: 16 c-MET binder A12 VH sequence

SEQ ID NO: 17 c-Met binder B10 VL sequence

SEQ ID NO: 18 c-MET binder B10 VH sequence

SEQ ID NO: 19 c-MET binder C10 VL sequence

SEQ ID NO: 20 c-MET binder C10 VH sequence

SEQ ID NO: 21 c-MET hinder E07 VL sequence

SEQ ID NO: 22 c-MET hinder E07 VH sequence

SEQ ID NO: 23 c-MET binder G02 VL sequence

SEQ ID NO: 24 c-MET binder G02 VH sequence

SEQ ID NO: 25 c-MET binder H06 VL sequence

SEQ ID NO: 26 c-MET binder H06 VH sequence

SEQ ID NO: 27 c-MET binder F03 VL sequence

SEQ ID NO: 28 c-MET binder F03 VH sequence

SEQ ID NO: 29 c-MET Binder F06 VL sequence

SEQ ID NO: 30 c-MET binder F06 VH sequence

SEQ ID NO: 31 c-MET binder B10v5 VL sequence

SEQ ID NO: 32 c-MET binder B10v5 VH sequence

SEQ ID NO: 33 c-MET binder CS06 VL sequence

SEQ ID NO: 34 c-MET binder CS06 VH sequence

SEQ ID NO: 35 glycine-serine linker

SEQ ID NO: 36 hinge 1

SEQ ID NO: 37 hinge 2

SEQ ID NO: 38 CL sequence

SEQ ID NO: 39 CH1 sequence

SEQ ID NO: 40 CH2 domain

SEQ ID NO: 41 CH3 domain (AG)

SEQ ID NO: 42 CH3 domain (GA)

SEQ ID NO: 43 humanized C225 VH S58R kinetic variant (hu225-L)

SEQ ID NO: 44 humanized C225 VL N108Y kinetic variant (hu225-M)

SEQ ID NO: 45 humanized C225 VH T109D kinetic variant (hu225-H)

SEQ ID NO: 46 humanized C225 VL N109E, T116N kinetic variant (hu225-H)

SEQ ID NO: 47 c-Met binder B10 VL variants comprising single or multipleamino acid substitutions

SEQ ID NO: 48 c-MET binder B10 VH kinetic variant Q6E (IMGT numbering)

SEQ ID NO: 49 c-MET binder F06 VL sequence variants comprising single ormultiple amino acid substitutions

SEQ ID NO: 50 c-Met binder F06 VL variants comprising single or multipleamino acid substitutions

SEQ ID NO: 51 c-Met binder B10v5 VL variants comprising single ormultiple amino acid substitutions

SEQ ID NO: 52 c-Met binder CS06 H kinetic variants

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention the term “comprise” encompasses the term “consist of”.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is entitled to antedate such disclosure byvirtue of prior invention.

The described objectives are solved by the present invention, preferablyby the subject matter of the appended claims. The inventors havesurprisingly found that heterodimeric bispecific immunoglobulinmolecules according to the invention can be used to overcome theresistance to EGFR- or c-MET-targeted monotherapies. In addition, theinventive heterodimeric bispecific immunoglobulin molecules havesurprisingly been found to bind cells which express one of EGFR or c-METwith a lower abundance with high selectivity.

The described objective is solved according to a first embodiment by theinventive heterodimeric bispecific immunoglobulin molecule whichcomprises

-   -   (i) a first Fab or scFv fragment which specifically binds to        EGFR, and    -   (ii) a second Fab or scFv fragment which specifically binds        c-MET, and    -   (iii) an antibody hinge region, an antibody CH2 domain and an        antibody CH3 domain comprising a hybrid protein-protein        interaction interface domain wherein each of said interaction        interface domain is formed by amino acid segments of the CH3        domain of a first member and amino acid segments of the CH3        domain of said second member, wherein said protein-protein        interface domain of the first chain is interacting with the        protein-protein-interface of the second chain by        homodimerization of the corresponding amino acid segments of the        same member of the immunoglobulin superfamily within said        interaction domains,

wherein the first or second engineered immunoglobulin chain has thepolypeptide sequence (“AG-SEED”):

(SEQ ID NO: 1) GQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPX₁DIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSKLTX₂DKSRWQQGNVFSCSVMHEALH NHYTQKX₃ISL,

wherein X₁, X₂ and X₃ may be any amino acid. For example, amino acidsrepresented by X₁, X₂ and X₃ may each independently from each other beselected from the group of naturally occurring amino acids. Engineeredimmunoglobulin chains which are comprised in the inventive heterodimericbispecific immunoglobulin molecule and the respective sequences thereofhave been described in WO 2007/110205. In the inventive heterodimericbispecific immunoglobulin molecule the term heterodimeric

A “heteromultimeric protein” according to the invention is a proteinmolecule comprising at least a first subunit and a second subunit,whereby each subunit contains a nonidentical domain. The inventiveheterodimeric bispecific immunoglobulin molecule comprises twonon-identical protein domains, e.g. “AG-SEED” and “GA-SEED” which willresult in a heterodimerization of the non-identical protein domains in aratio of 1:1. The inventive heterodimeric bispecific immunoglobulinmolecule according to a first embodiment comprises a first Fab or scFvfragment which specifically binds to EGFR. The term Fab fragment refersto an antigen binding antibody fragment which can e.g. be obtained bypapain treatment of IgG type immunoglobulins, which will result in twoFab fragment and an Fc domain. Functional aspects and pmethods to obtainFab fragments are described e.g. in “Applications and Engineering ofMonoclonal Antibodies” by D. J. King, CRC Press, 1998, chapter 2.4.1Zaho et al, Protein Expression and Purification 67 (2009) 182-189; S. M.Andrew, J. A. Titus, Fragmentation of immunoglobulin G, Curr. Protoc.Cell Biol. (2003) Unit 16.14 (Chapter 16). The inventive heterodimericbispecific immunoglobulin molecule may e.g. also comprise a first scFvfragment that specifically binds to EGFR, The term “scFv” as used in thepresent invention refers to a molecule comprising an antibody heavychain variable domain (or region; VH) and an antibody light chainvariable domain (or region; VL) connected by a linker, and lacksconstant domains, e.g. an scFv fragment according to the invention maye.g. include binding molecules which consist of one light chain variabledomain (VL) or portion thereof, and one heavy chain variable domain (VH)or portion thereof, wherein each variable domain (or portion thereof) isderived from the same or different antibodies. scFv molecules preferablycomprise an linker interposed between the VH domain and the VL domain,which may e.g. include a peptide sequence comprised of the amino acidsglycine and serine. For example, the peptide sequence may comprise theamino acid sequence (Gly₄ Ser)_(n), whereby n is an integer from 1-6,e.g. n may be 1, 2, 3, 4, 5, or 6, preferably n=4 scFv molecules andmethods of obtaining them are known in the art and are described, e.g.,in U.S. Pat. No. 5,892,019, Ho et al. 1989. Gene 77:51; Bird et al. 1988Science 242:423: Pantoliano et al. 1991. Biochemistry 30:10117; Milenicet al. 1991. Cancer Research 51:6363; Takkinen et al. 1991. ProteinEngineering 4:837.

A first Fab or scFv fragment of the inventive heterodimeric bispecificimmunoglobulin molecule specifically binds to human epidermal growthfactor receptor (EGFR). Specific binding, or any grammatical variantthereof, refers to a binding of the first Fab or scFv fragement with anKd of at least 1×10⁻⁵ M, e.g. 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M,1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² to EGFR. EGFR according to the inventionrefers to EGFR having the sequences as provided by UniProtKB databaseentry P00533, including all of its isoforms and sequence variants(UniProtKB database entries P00533-1, P00533-2, P00533-3, P00533-4), orany of the mutations described in Cai et al., PLoS ONE 9(4): e95228,such as e.g. c.2126A>C, c.2155G>T, c.2156G>C, c.2235_2249del15,c.2236_2250del15, c.2237_2251del, c. 2239_2248ATTAAGAGGAG>C,c.2240_2257del 18, c2248G>C, c.2303G>T, c.2573T>G, c.2582T>A,p745del_frameshift, p.L858R, p.S768I.

The inventive heterodimeric bispecific immunoglobulin molecule furthercomprises a second Fab or scFv fragment which specifically binds toc-MET. c-MET as used herein refers to MET Proto-Oncogene, ReceptorTyrosine Kinase (UniProtKB database antry P08581), which may also bereferred to as Hepatocyte Growth Factor Receptor. For example, c-METalso includes sequence variants such as those disclosed in Nat Genet.1997 May; 16(1)68-73, e.g. c-MET R970C (MET^(R970C)) c-MET T992I(MET^(T922I)), MET^(M1149T), MET^(V1206L), MET^(V1238I), MET^(D1246N),MET^(Y1248C), MET^(C1213V), MET^(D1246H), MET^(Y1248H), MET^(M1268T),MET^(A320V), MET^(N375S). Specific binding of the second Fab or scFvfragment to c-MET refers to a binding of the second Fab or scFvfragement with an K_(d) of at least 1×10⁻⁵ M, e.g. 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² to c-MET.

The inventive heterodimeric bispecific immunoglobulin molecule accordingto a first embodiment of the invention further comprises antibody hingeregion, an antibody CH2 domain and an antibody CH3. For example, thereare five classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM) all ofwhich contain a hinge region and which may be comprised in the inventiveheterodimeric bispecific immunoglobulin molecule. Additionally, some ofthese classes of immunoglobulins have subclasses, e.g. IgG has foursubclasses (IgG1, IgG2, IgG3, and IgG4), (Alberts, B et al., Chapter 23:The Immune System, In Molecular Biology of the Cell, 3d Edition, GarlandPublishing, Inc., New York, N.Y.), the hinge regions of which may alsobe comprised in the heterodimeric bispecific immunoglobulin molecule ofthe invention. The hinge region may e.g. be divided into three regions:the upper, middle, and lower hinge. The upper hinge is defined as thenumber of amino acids between the end of the first domain of the heavychain (CH1) and the first cysteine forming an inter heavy chaindisulfide bridge. The middle hinge is high in proline and contains theinter-heavy chain cysteine disulfide bridges. The tower hinge connectsthe middle hinge to the CH2 domain (see e.g. Sandie, I. and Michaelsen,T., Chapter 3: Engineering the Hinge Region to OptimizeComplement-induced Cytolysis, In Antibody Engineering: A PracticalGuide, W. H. Freeman and Co, New York, N.Y.; Hamers-Casterman, C.,Naturally Occurring Antibodies Devoid of Light Chains, 363 Nature 446(1993) and Terskikh, A. V., “Peptabody”: A New Type of High AvidityBinding Protein, 94 Proc. Natl. Acad. Sci. USA 1663 (1997)). The hingeregion of the inventive inventive heterodimeric bispecificimmunoglobulin molecule may e.g. also comprise any of the amino acidsequences of the hinge regions disclosed in J. of Biological Chem. VOL.280, NO. 50, pp. 41494-41503, Dec. 16, 2005.

In one embodiment, the heterodimeric bispecific immunoglobulin moleculeof the invention comprises as first member IgG of the immunoglobulinsuper family and as second member IgA. For example, the inventiveheterodimeric bispecific immunoglobulin molecule may in one embodimentcomprise the hinge region according to the amino acid sequence of SEQ IDNO: 1, or SEQ ID NO: 2. For example, the inventive heterodimericbispecific immunoglobulin molecule may comprise derivatives of human IgGand IgA CH3 domains which create complementary human strand-exchangeengineered domain (SEED) CH3 heterodimers that are composed ofalternating segments of human IgA and IgG CH3 sequences as described inProtein Engineering, Design & Selection vol. 23 no. 4 pp. 195-202, 2010WO 2007/110205 A1). The resulting pair of SEED CH3 domainspreferentially associates to form heterodimers when expressed inmammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1hinge]-CH2-[SEED CH3].

In one embodiment the heterodimeric bispecific immunoglobulin moleculeof the invention as disclosed above comprises a first or secondengineered immunoglobulin chain (“AG-SEED”) which has the polypeptidesequence according to SEQ ID NO:2 in which X₁ is K or S, X₂ is V or T,and X₃ is T or S. For example, the first or second engineeredimmunoglobulin chain of the of the inventive heterodimeric bispecificimmunoglobulin molecule may comprise an amino acid sequence according toSEQ ID NO: 2 in which X₁ is K, X₂ is V, and X₃ is S. X₁ is K, X₂ is V,and X₃ is T, X₁ is K, X₂ is T, and X₃ is S, X₁ is K, X₂ is T, and X₃ isT. X₁ is S, X₂ is V, and X₃ S, X₁ is S, X₂ is V, and X₃ is T, X₁ is S,X₂ is T, and X₃ is S, or X₁ is S, X₂ is T, and X₃ is T.

In one embodiment the inventive heterodimeric bispecific immunoglobulinmolecule as disclosed above comprises a first or second engineeredimmunoglobulin chain which has the polypeptide sequence according to SEQID NO: 3 (“GA-SEED”), whereby wherein X₁, X₂, X₃, X₄, X₅ and X₆ may beany amino acid, e.g. X₁, X₂, X₃, X₄, X₅, X₆ may be independentlyselected from alanine, arginine, asparagine, aspartic acid, asparagineor aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, or valine. Accordingto a one embodiment, the first or second engineered immunoglobulin chainof the inventive heterodimeric bispecific immunoglobulin molecule asdisclosed above has the amino acid sequence according to SEQ ID NO:3,wherein X₁ is L or Q, X₂ is A or T, X₃ is L, V, D or T; X₄ is F, A, D,E, G, H, K, N, P, Q, R, S or T; X₅ is A or T, and X₆ is E or D. In apreferred embodiment, the first engineered immunoglobulin chaincomprises the amino acid sequence according to SEQ ID NO: 5 (“AG-SEED”)and the second engineered immunoglobulin chain of the inventiveheterodimeric bispecific immunoglobulin molecule as disclosed abovecomprises the amino acid sequence according to SEQ ID NO: 6 (“GA-SEED”).

In one embodiment the inventive heterodimeric bispecific immunoglobulinmolecule binds to EGFR as disclosed above with an affinity of at LeastK_(D)=5×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² to EGFR.According to one embodiment the inventive heterodimeric bispecificimmunoglobulin molecule binds to c-MET as disclosed above with anaffinity of at least K_(D)=5×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M,1×10³¹ ¹² to c-MET. For example, the heterodimeric bispecificimmunoglobulin molecule of the invention as disclosed above binds via afirst and second Fab or scFv fragment c-MET and EGFR with an affinity ofK_(D)=5×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M. EGFR andc-Met may e.g. be present on a single cell, such as a cancer cell, ore.g. to a cell, such as e.g. cancer cell, which may be single cell, apluarality of cells, or tumor tissue that expresses both c-MET and EGFR.The cells may e.g. also be in suspension, or detached from tissue andmay circulate in the blood stream of an individual, such as a humaninflicted with cancer. For example, the affinity of first and second Faband/or scFv fragments of the inventive heterodimeric bispecificimmunoglobulin molecule may be determined by ELISA, or surface plasmonresonance as described in J. Biochem. Biophys. Methods 57 (2003)213-236, Current Protocols in Protein Science (2006) 19.14.1-19.14.17.

According to one embodiment the first Fab or scFv fragment of theheterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above is derived from cetuximab (C225). For example, the firstFab or scFv fragment of the heterodimeric bispecific immunoglobulinmolecule may comprise VL and VH sequences of cetuximab, or e.g. VL andVH sequences of cetuximab which have been humanized. For example,humanized as used for the inventive heterodimeric bispecificimmunoglobulin molecule refers to a chimeric antibody or antibodyfragment which contain minimal sequence derived from non-humanimmunoglobulin. Humanization of a given antibody sequence will result ina reduction of the immunogenicity of a xenogenic antibody, such as amurine antibody, or chimeric antibody which already comprises humansequences, for introduction into a human, while maintaining the fullantigen binding affinity and specificity of the antibody. For example,cetuximab is a chimeric antibody which is composed of the Fv (variable;antigen-binding) regions of the 225 murine EGFR monoclonal antibodyspecific for the N-terminal portion of human EGFR with human IgG1 heavyand kappa light chain constant (framework) regions.

Humanization may e.g. comprise CDR grafting technology which involvessubstituting the complementarity determining regions of, for example, amouse antibody, into a human framework domain, e.g., see WO 02/22653.Strategies and methods for the resurfacing of antibodies, and othermethods for reducing immunogenicity of antibodies within a differenthost, are disclosed in U.S. Pat. No. 5,639,641. Antibodies can behumanized using a variety of other techniques including CDR-grafting(see e.g. EP 0 239 400 B1; WO 91/09967; U.S. Pat. Nos. 5,530,101;5,585,089), veneering or resurfacing (see e.g. EP 0 592 106; EP 0 519596; Padlan E. A., 1991, Molecular Immunology 28(4/5); 489-498;Studnicka G. M. et al., 1994, Protein Engineering, 7(6): 805-814;Roguska M. A. et al., 1994. PNAS, 91: 969-973), chain shuffling (seee.g. U.S. Pat. No. 5,565,332), and identification of flexible residues(see e.g. WO2009032661). Human antibodies can be made by a variety ofmethods known in the art including phage display methods, such as .g.U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; andinternational patent application publication numbers WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, WO91/10741. Accordingly, the first Fab or scFv fragment of theheterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above may comprise VL and VH sequences according to to any oneof SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:43, SEQ ID NO. 44, SEQ ID NO: 45, SEQ ID NO: 46. For example, the VLamino acid sequence of first Fab or scFv fragment of the heterodimericbispecific immunoglobulin molecule may comprise the amino acid sequenceaccording to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 44, SEQ ID NO: 46and VH amino acid sequences selected from SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 11, SEQ ID NO: 12. VL and VH sequences of the first Fab orscFv fragment as disclosed above may e.g. comprise SEQ ID NO:43 and SEQID NO: 9, SEQ ID NO: 44 and SEQ ID NO: 9, or SEQ ID NO: 45 and SEQ IDNO: 9, or e.g. SEQ ID NO: 43 and SEQ ID NO: 9, or SEQ ID NO:45 and SEQID NO: 9, or SEQ ID NO: 46 and SEQ ID NO: 11.

According to one embodiment the second Fab or scFv fragment of theinventive heterodimeric bispecific immunoglobulin molecule has disclosedabove comprises VL sequences selected from the group consisting of SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51.

In one embodiment the the second Fab or scFv fragment of the inventiveheterodimeric bispecific immunoglobulin molecule has disclosed abovecomprises VH sequences selected from the group consisting of SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 48, ,SEQ ID NO: 50, or SEQ ID NO: 52.

According to one embodiment, the first and second Fab or scFv fragmentsof the heterodimeric bispecific immunoglobulin molecule of the inventionas disclosed above comprise the amino acid sequences selected from SEQID NO: 9, SEQ ID NO: 43, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 47,SEQ ID NO: 48, (e.g. which may be comprised in the inventive molecule“225-LxB10”), or SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 31, SEQ ID NO:51, SEQ ID NO:32 (e.g. which may be comprised in the inventive molecule“225-MxB10v5”), or SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ IDNO: 49, SEQ ID NO: 30, SEQ ID NO: 50, (e.g. which may be comprised inthe inventive molecule “225-HxF06”), or SEQ ID NO: 45, SEQ ID NO: 46,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52 (e.g. which may be comprisedin the inventive molecule “225-HxCS06”), or SEQ ID NO: 11, SEQ ID NO:44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52 (e.g. which may becomprised in the inventive molecule “225-MxCS06”).

According to one embodiment the first and second Fab or scFv fragmentsof the heterodimeric bispecific immunoglobulin molecule of the inventionas disclosed above comprise the amino acid sequences according to SEQ IDNO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO; 52 (e.g.corresponding to the inventive molecule “225-MxCS06”), or SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO:50 (e.g. corresponding to the inventive molecule “225-HxCS06”).

In one embodiment the inventive the Fc domain of the heterodimericbispecific immunoglobulin molecule interacts with the neonatal Fcreceptor (FcRn). FcRn is a major histocompatibility complex class I-likeheterodimer composed of the soluble light chain β2-microglobulin (β2m)and a membrane-bound heavy chain. Crystal structure analysis revealedthat the human FcRn (hFcRn) binds to the CH2-CH3 hinge region of bothheavy chains of the Fc homodimer of an IgG, resulting in a 2:1stoichiometry. The interaction between FcRn and Fc is mainly stabilizedby salt bridges between anionic FcRn residues and histidine residues ofthe IgG, which are protonated at acidic pH. Site-directed mutagenesisstudies and crystal structure analysis of the FcRn/IgG Fc complex showthat the Fc amino acid residues at positions 252-256 in the CH2 domainsand at 310, 433, 434, and 435 in the CH3 domains are at the core or inclose proximity to the FcRn interaction site, and that the conservedhistidine residues H310 and possibly H435 are responsible for the pHdependence (see e.g. mAbs 6:4, 928-942, July/August 2014; Nature ReviewsImmunology 7, 715-725 (September 2007)). For example, the inventiveheterodimeric bispecific immunoglobulin molecule may interact with theFcRn via salt bridges as disclosed above, or may interact with FcRn bysalt bridges that involve other amino acids of both AG-SEED and GA-SEED,thereby protecting the inventive heterodimeric bispecific immunoglobulinmolecule from degradation and extending its serum half-life. Extendedhalf-life of the inventive heterodimeric bispecific immunoglobulinmolecule may e.g. be employed to minimize adverse reactions caused byhigh doses of the inventive heterodimeric bispecific immunoglobulinmolecule if administered to an individual e.g. by i v. or i.m.application, which will e.g. also result in a decreased frequency ofinjection of the inventive heterodimeric bispecific immunoglobulinmolecule. This will e.g. also reduce the financial burden on anindividual which may be in need of a treatment with the inventiveheterodimeric bispecific immunoglobulin molecule. For example, sequencevariants or the AG-SEED and GA-SEED may be used to reduce theinteraction of the inventive heterodimeric bispecific immunoglobulinmolecule with FcRn thereby shortening its serum half-life. Sequencevariants e.g. include those disclosed above, AG-SEED with X₁, X₂ and X₃representing any amino acid, or e.g. preferably an AG-SEED in which X₁is K or S, X₂ is V or T, and X₃ is T or S, e.g. a GA-SEED as disclosedabove wherein X₁, X₂, X₃, X₄, X₅ and X₆ may be any amino acid. It maye.g. be preferred that in the GA-SEED X₁ is L or Q, X₂ is A or T, X₃ isL, V, D or T, X₄ is F, A, D, E, G, H, K, N, P, Q, R, S or T; X₅ is A orT, and X₆ is E or D.

In one embodiment, the amino acids of the inventive heterodimericbispecific immunoglobulin molecule as disclosed above which interactwith FcRn are derived from IgG1, preferably human IgG1. For example, theamino acids which interact with FcRn comprise those of wildtype IgG1 asdisclosed above, e.g. Fc amino acid residues at positions 252-256 in theCH2 domains and at 310, 433, 434, and 435 in the CH3 domains are at thecore or in close proximity to the FcRn interaction site, whereby theconserved histidine residues H310 and possibly H435 may e.g. confer forthe pH dependence of the interaction between the inventive heterodimericimmunoglobulin molecule and FcRn.

In one embodiment the inventive heterodimeric bispecific immunoglobulinmolecule as disclosed above mediates antibody-dependent cellularcytotoxicity. For example, the inventive heterodimeric bispecificimmunoglobulin molecule induces ADCC when bound to EGFR and c-METexpressed on the surface of the same cell cell, or e.g. when bound totwo cells, one of which expresses EGFR and the second one of whichexpresses c-MET, whereby e.g. EGFR and c-Met are as defined above.Binding of the heterodimeric bispecific immunoglobulin molecule of theinvention to EGFR and c-Met present on the same cell or on twoindividual cells, but preferably one the same cell, is as disclosedabove. The term ADCC (antibody dependent cell cytotoxicity) as used forthe inventive heterodimeric bispecific immunoglobulin molecule refers toa mechanism of cell-mediated immune defense whereby an effector cell ofthe immune system actively lyses a target cell, whose membrane-surfaceantigens have been bound by specific antibodies. ADCC is mediated bye.g. the binding of CD16 (FcyRIII) expressed on NK cells to the Fcdomain of antibodies (see e.g. Clynes et al. (2000) Nature Medicine 6,443-446). ADCC may e.g. be improved by amino acid substitutions in theFc domain which affect the binding of the Fc domain to CD16. Forexample. Shields et al. (J Biol Chem 9(2), 6591-6604 (2001)) showed thatamino acid substitutions at positions 298, 333, and/or 334 of the Fcregion (EU numbering of residues) improve ADCC. Alternatively, increasedFc receptor binding and effector function may e.g. be obtained byaltering the glycosylation of the Fc region. The two complex biantennaryoligosaccharides attached to Asn 297 of the Fc domain are typicallyburied between the CH2 domains, forming extensive contacts with thepolypeptide backbone, and their presence is essential for the antibodyto mediate effector functions including ADCC (Lifely et al.,Glycobiology 5, 813-822 (1995); Jefferis et al., Immunol Rev 163, 59-76(1998); Wright and Morrison, Trends Biotechnol 15, 26-32 (1997)),Overexpression of e.g. β(1,4)-N-acetylglucosaminyltransferase III(GnTIII), a glycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofantibodies. Thus overexpression of e.g. of GnTIII in cell lines used forthe production of the inventive heterodimeric bispecific immunoglobulinmolecule, may result in inventive fusion proteins enriched in bisectedoligosaccharides, which are generally also non-fucosylated and mayexhibit increased ADCC.

In one embodiment the invention provides an isolated polynucleotide whenencodes at least one of the amino acid sequences according to SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDSEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO. 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ IDNO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQID NO:40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO:44, SEQID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49, SEQID NO: 50, SEQ ID NO:51, SEQ ID NO: 52 of the inventive bispecificheterodimeric immunoglobulin molecule. For example, the isolatedpolynucleotide of the invention may encode at least one, e.g. one, two,three, four, five, six, seven, eight, nine or ten of the amino acidsequences as disclosed above. For example, in one embodiment theisolated polynucleotide comprises polynucleotides which encode at leastone of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO. 7,SEQ ID NO: 8, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ IDNO: 52 of the inventive bispecific heterodimeric immunoglobulinmolecule. For example the isolated polynucleotide of the invention maycomprise polynucleotides which encode amino acid sequences according to(225M, CS06) SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34,SEQ ID NO: 52, or (225H, CS06) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50. For example, in oneembodiment the isolated polynucleotide according to the invention maye.g. comprise polynucleotides encoding the amino acid sequencesaccording to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO:44,SEQ ID NO:45, SEQ ID NO:46. In one embodiment the polynucleotideaccording to the invention e.g. comprises polynucleotides which encodethe amino according to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:45, SEQID NO:46. For example, in one embodiment the inventive polynucleotideencodes amino acid sequences according to SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO:45, SEQ ID NO:46. In one embodiment, the polynucleotideaccording to the invention comprises polynucleotides which encode theamino acid sequences selected from SEQ ID NO: 9, SEQ ID NO: 43, SEQ IDNO: 17, SEQ ID NO:18, or SEQ ID NO: 47, SEQ ID NO: 48 (e.g. which may becomprised in the inventive molecule “225-LxB10”), or SEQ ID NO: 11, SEQID NO: 44, SEQ ID NO: 31, SEQ ID NO: 51, SEQ NO:32 (e.g. which may becomprised in the inventive molecule “225-MxB10v5”), or SEQ ID NO:45, SEQID NO: 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50,(e.g. which may be comprised in the inventive molecule “225-HxF06”), orSEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:52 (e.g. which may be comprised in the inventive molecule “225-HxCS06”),or SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ ID NO: 34, SEQ IDNO: 52 (e.g. which may be comprised in the inventive molecule“225-MxCS06”), or SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 52 (e.g. corresponding to the inventive molecule“225-MxCS06”), or SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO:49, SEQ ID NO: 30, SEQ ID NO: 50 (e.g. corresponding to the inventivemolecule “225-HxCS06”), or (225M, CS06) SEQ ID NO: 11, SEQ ID NO: 44,SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 52, or (225H, CS06) SEQ IDNO:45, SEQ ID NO: 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQID NO 50, or (e.g. corresponding inventive molecule “225M, B10v5”) SEQID NO: 11, SEQ ID NO: 44, SEQ ID NO: 31, SEQ ID NO: 51, SEQ ID NO:32, or(e.g. corresponding inventive molecule “225H, CS06”) SEQ ID NO:45, SEQID NO: 46, SEQ ID NO: 29, SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 50.For example, the nucleotide sequence of each of the above amino acidsequences of the invention may be obtained by translation usingweb-based tools, such as “Translate tool”(http://web.expasy.org/translate/) and may e.g. be codon-optimizedaccordance with the intended expression system or host (see e.g. TrendsMol Med. 2014 November; 20(11):604-13; Genome Res. 2007 April; ,17(4):401-4). For example, the polynucleotides encoding the amino acidsequences as disclosed above may be comprised on individualpolynucleotides, each of which is considered a polynucleotide accordingto the invention, or e.g. the polynucleotide according to the inventionmay comprise polynucleotides encoding two of the amino acid sequences asdisclosed above e.g. SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, SEQID NO: 34, or SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO: 29, SEQ ID NO:30, or SEQ ID NO: 11, SEQ ID NO: 9, or SEQ ID NO 49, SEQ ID NO: 50. Thepolynucleotides according to the invention as disclosed above may e.g.be used for the production of the inventive bispecific heterodimericimmunoglobulin molecule, e.g. by heterologous expression in a suitablehost, or host cell.

The term “isolated” as used with the polynucleotides according to theinvention refers to polynucleotides which are separated from e.g.constituents, cellular and otherwise, in which the polynucleotide arenormally associated with in nature, e.g. the isolated polynucleotide isat least 80%, 90%, 95% pure by weight, devoid or contaminatingconstituents. For example, isolated polynucleotides of the invention mayrefer to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated polynucleotide” may comprise a DNA molecule inserted intoa vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a procaryote or eucaryote.

In one embodiment the present invention provides a vector whichcomprises at least one polynucleotide according to the invention asdisclosed above. The tem vector or expression vector according to theinvention refers to a nucleic acid molecule capable of extra-chromosomalreplication. Preferred vectors are those capable of autonomousreplication and expression of nucleic acids to which they are linked.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of “plasmids” which refer generally to circular doublestranded DNA loops which, in their vector form are not bound to thechromosome. Nucleic acid sequences necessary for expression of theheterodimeric bispecific immunoglobulin molecule in eukaryotic cellscomprise e.g. at least one promoter, and enhancers, termination andpolyadenylation signals as well as a selectable marker, such as e.g. anantibiotic resistance. Expression vectors which may be used forexpression of the inventive heterodimeric bispecific immunoglobulinmolecule may e.g. comprise pCMV, pcDNA, p4X3, p4X4, p4X5, p4X6, pVL1392,pVL1393, pACYC177, PRS420, or if viral based vector systems are to beused e.g. pBABEpuro, pWPXL, pXP-derived vectors may e.g. comprise pCMV,pcDNA, p4X3, p4X4, p4X5, p4X6, pVL1392, pVL1393, pACYC177, PRS420, or ifviral based vector systems are to be used e.g. pBABEpuro, pWPXL,pXP-derived vectors.

In one embodiment, present invention provides a host cell whichcomprises the polynucleotide sequence or vector as disclosed above, e.g.a polynucleotide or vector or expression vector which comprises at leastone coding sequence for the inventive heterodimeric bispecificimmunoglobulin molecule as disclosed above. For example, a host cell foruse in the invention may be a yeast cell, insect cell or mammalian cell.For example, the host cell of the invention may be an insect cellselected from Sf9, Sf21, S2, Hi5, or BTI-TN-5B1-4 cells, or e.g. thehost cell of the invention may be a yeast cell selected fromSaccharomnyces cerevislae, Hansenula polymorpha, Schizosaccharomycespombe, Schwanniomyces occidentalis, Kluyveromyceslactis, Yarrowialipolytica and Pichia pastoris, or e.g. the host cell of the inventionmay be a mammalian cell selected from HEK293, HEK293T, HEK293E, HEK293F, NS0, per.C6, MCF-7, HeLa, Cos-1, Cos-7, PC-12, 3T3, Vero, vero-76,PC3, U87, SAOS-2, LNCAP, DU145, A431, A549, B35, H1299, HUVEC, Jurkat,MDA-MB-231, MDA-MB-468, MDA-MB-435, Caco-2, CHO, CHO-K1, CHO-B11,CHO-DG44, BHK, AGE1.HN, Namalwa, WI-38, MRC-5, HepG2, L-929, RAB-9,SIRC, RK13, 11B11, 1D3, 2.402, A-10, B-35, C-6, F4/80, IEC-18, L2,MH1C1, NRK, NRK-49F, NRK-52E, RMC, CV-1, BT, MDBK, CPAE, MDCK.1, MDCK.2,and D-17.

In one embodiment the invention provides a method for producing theheterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above, whereby the inventive method comprises the steps ofculturing a host cell according to the invention as disclosed aboveunder conditions sufficient for the heterologous expression of saidheterodimeric bispecific immunoglobulin molecule and purifying saidheterodimeric bispecific immunoglobulin molecule. For example, hostcells of the invention may be allowed to grow in DMEM containing 10%FBS, and were incubated at 37° C. in 10% CO₂ or e.g. in protein-freeculture medium to aid in the subsequent isolation and purification, ore.g. in Grace's insect medium, express Five ® SFM (Life Technologies),or High Five® medium (Life Technologies), YNM medium, YPD broth, or e.g.PichiaPink (Life technologies). For example, expression of theinventive, heterodimeric bispecific immunoglobulin molecule in mammaliancells may be done according to the method as described in Methods MolBiol. 2012; 907:341-58. Insect cells may e.g. also be used for theexpression of the inventive heterodimeric bispecific immunoglobulinmolecule such as e.g. Drosophila S2 cells as described in Journal ofImmunological Methods 318 (2007) 37-46. Yeast cells, for example, mayalso be used for the expression of the inventive heterodimericbispecific immunoglobulin molecule, such as Pichia pastoris as describedin Appl Microbiol Biotechnol. 2014 December; 98(24):10023-39, orBiotechnol Lett. 2015 July; 37(7):1347-54.

The host cells of the invention may e.g. be allowed to grow between12-408 h, e.g. for about 12 to about 400 h, e.g. between 14 h, 16 h, 18h, 20 h, 24 h, 36 h, 48 h, 72 h, 96 h to about 120 h, 144 h, 168 h, 192,216 h, 240 h, 264 h, 288 h, 312 h, 336 h, 360 h, 384 h, 408 h.Subsequently, the inventive vNAR or inventive fusion protein may beisolated and purified. For example, the heterodimeric bispecificimmunoglobulin molecule of the invention may be purified and isolated bychromatography, e.g. ion-exchange chromatography, size-exclusionchromatography, ammonium sulfate precipitation, or ultrafiltration. Forexample, the inventive heterodimeric bispecific immunoglobulin moleculemay also comprise a signal sequence, which refers to an amino acidsequence which is capable of initiating the passage of a polypeptide, towhich it is operably linked, e.g. by a peptide bond, into theendoplasmic reticulum (ER) of a host cell. The signal peptide isgenerally cleaved off by an endopeptidase (e.g. a specific ER-locatedsignal peptidase) to release the (mature) polypeptide. The length of asignal peptide is typically in the range from about 10 to about 40 aminoacids.

In one embodiment the invention provides a heterodimeric bispecificimmunoglobulin molecule according to the invention as disclosed abovewhich is obtainable by the inventive method as disclosed above. Forexample, the heterodimeric bispecific immunoglobulin molecule of theinvention as disclosed above may be produced by the inventive method asdisclosed above and isolated.

In one embodiment the heterodimeric bispecific immunoglobulin moleculeof the invention as disclosed above is covalently coupled to at leastone linker. The term “linker” or “linker peptide” refers to a syntheticor artifical amino acid sequence that connects or links two molecules,such as e.g. two polypeptide sequences that link two polypeptidedomains, or e.g. a protein and a cytostatic drug, or toxin. The term“synthetic” or “artifical” as used in the present invention refers toamino acid sequences that are not naturally occurring. The linker whichis covalently bound to the heterodimeric bispecific immunoglobulinmolecule of the invention is cleavable or non-cleavable. The term“cleavable” as used in the present invention refers to linkers which maybe cleaved by proteases, acids, or by reduction of a disulfide body(e.g. glutathion-mediated or glutathion sensitive). For example,cleavable linkers may comprise valine-citrulline linkers, hydrazonelinkers, or disulfide linkers. Non-cleavable linkers which may e.g. becovalently bound to the amino donor-comprising substrate of theinvention comprise maleimidocaproyllinker to MMAF (mc-MMAF),N-maleimidomethylcyclohexane-1-carboxylate MCC), ormercapto-acetamidocaproyl linkers. For example, the linkers which arecovalently coupled to the inventive heterodimeric bispecificimmunoglobulin molecule may also include linkers as described in WO2010/138719, or e.g. those described in WO 2014/093379.

In one embodiment the linker of the heterodimeric bispecificimmunoglobulin molecule of the invention as disclosed above is coupledto a dye, radioisotope, or cytotoxin, The term “coupled” as used for thelinker as disclosed above refers to the fact that the dye, radioisotopeor cytoxin may e.g. be non-covalently via e.g. ionic, or hydrophobicinteractions, or covalently attached to the linker molecule as disclosedabove. For example, the linker may comprise streptavidin and the dye,radioisotope or cytotoxin may be covalently bound to biotin. Forexample, the dye which may be covalently linked or coupled to theinventive heterodimeric bispecific immunoglobulin molecule may also be afluorophore, such as e.g. 1,8-ANS, 4-methylumbelliferone,7-amino-4-methylcoumarin, 7-hydroxy-4-methylcoumarin, Acridine, AlexaFluor 350™, Alexa Fluor 405™, AMCA, AMCA-X, ATTO Rho6G, ATTO Rho11, ATTORho12, ATTO Rho13, ATTO Rho14, ATTO Rho101, Pacific Blue, Alexa Fluor430™, Alexa Fluor 480™, Alexa Fluor 488™, BODIPY 492/515, Alexa Fluor532™, Alexa Fluor 546™, Alexa Fluor 555™, Alexa Fluor 594™, BODIPY505/515, Cy2, cyQUANT GR, Fluo-3, Fluo-4, GFP (EGFP), mHoneydew, OregonGreen™ 488, Oregon Green™ 514, EYFP, DsRed, DsRed2, dTomato, Cy3.5,Phycoerythrin (PE), Rhodamine Red, mTangerine, mStrawberry, mOrange,mBanana, Tetramethylrhodamine (TRITC), R-Phycoerythrin, ROX, DyLight594, Calcium Crimson, Alexa Fluor 594™, Alexa Fluor 610™, Texas Red,mCherry, mKate, Alexa Fluor 660™, Alexa Fluor 680™ allophycocyanin,DRAQ-5, carboxynaphthofluorescein, C7, DyLight 750, Cellvue NIR780,DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes(IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue,Methoxy coumarin, Naphtho fluorescein, PyMPO,5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxyrhodamine, 6- carboxyrhodamine, 6-carboxytetramethyl amino,Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, HEX, 6-JOE, NBD(7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500,Oregon Green 514, Pacific Blue, phthalic acid, terephthalic add,isophthalic acid, cresyl fast violet, cresyl blue violet, brilliantcresyl blue, para-aminobenzoic acid, erythrosine, phthalocyanines,azomethines, cyanines, xanthines, succinylfluoresceins, rare earth metalcryptales, europium trisbipyridine diamine, a europium cryptate orchelate, diamine, dicyanins, or La Jolla blue dye. Dyes which may beused in the invention may e.g. also include quantum dots. The termquantum dot as used in the present invention refers to a singlespherical nanocrystal of semiconductor material where the radius of thenanocrystal is less than or equal to the size of the exciton Bohr radiusfor that semiconductor material (the value for the exciton Bohr radiuscan be calculated from data found in handbooks containing information onsemiconductor properties, such as the CRC Handbook of Chemistry andPhysics, 83rd ed., Lide, David R. (Editor), CRC Press, Boca Raton, Fla.(2002)). Quantum dots are known in the art, as they are described inreferences, such as Weller, Angew. Chem. Int. Ed. Engl. 32: 41-53(1993), Alivisatos, J. Phys. Chem. 100: 13226-13239 (1996), andAlivisatos, Science 271: 933-937 (1996), Quantum dots may e.g. be fromabout 1 nm to about 1000 nm diameter, e.g. 10 nm, 20 nm, 30 nm, 40 nm,50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300nm, 350 nm, 400 nm, 450 nm, or 500 nm, preferably at least about 2 nm toabout 50 nm, more preferably QDs are at least about 2 nm to about 20 nmin diameter (for example about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nm). QDs are characterized by theirsubstantially uniform nanometer size, frequently exhibitingapproximately a 10% to 15% polydispersion or range in size. A QD iscapable of emitting electromagnetic radiation upon excitation (i.e., theQD is photoluminescent) and includes a “core” of one or more firstsemiconductor materials, and may be surrounded by a “shell:” of a secondsemiconductor material, A QD core surrounded by a semiconductor shell isreferred to as a “core/shell” QD. The surrounding “shell” material willpreferably have a bandgap energy that is larger than the bandgap energyof the core material and may be chosen to have an atomic spacing closeto that of the “core” substrate. The core and/or the shell can be asemiconductor material including, but not limited to, those of thegroups II-VI (ZnS, ZnSe, ZnTe, US, CdSe, CdTe, HgS, HgSe, HgTe, MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and thelike) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and thelike) and IV (Ge, Si, and the like) material, PbS, PbSe, and an alloy ora mixture thereof. Preferred shell materials include ZnS. Quantum dotsmay be coupled to the inventive linker, enzyme, or protein by any methodknown in the art such as e.g. the methods disclosed Nanotechnology 2011Dec. 9; 22(49):494006; Colloids and Surfaces B: Biointerfaces 84 (2011)360-368. For example, the linker as disclosed above may be covalentlybound or coupled to a radioisotope such as e.g. ⁴⁷Ca, ¹⁴C, ¹³⁷Cs, ⁵⁷Co,⁶⁰Co, ⁸⁷CU, ⁸⁷Ga, ¹²³I, ¹²⁵I, ¹²⁹I, ¹³¹I, ³²P, ⁷⁵Se, ⁸⁵Sr, ³⁵S, ²⁰¹Th,³H, preferably, the radioisotopes are incorporated into a furthermoelcule, such as e.g. a chelator. Typical chelators that may e.g. beused as a further molecule covalently bound to the aminodonor-comprising substrate of the invention are DPTA, EDTA(Ethylenediamine-tetraacetic acid), EGTA(Ethyleneglycol-O,O′-bis(2-aminoethyl-N,N,N′,N′-tetraacetic acid, NTA(Nitrilotriacetic acid), HEDTA(N-(2-Hydroxyethyl)-ethylenediaminee-N,N′,N′-triaceticacid), DTPA(2-[Bis[2-[bis(carboxymethyl)amino]-ethyl]amino]acetic acid), or DOTA(1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid).

For example, the linker may be covalently coupled to a cytotoxin, whichmay e.g. also be referred to as “payload” (see e.g. Perez et al. DrugDiscovery Today Vol 19 (7), July 2014). Cytotoxins which are e.g. suitedfor covalent attachment to linker molecules may be grouped into two mainclasses: The first class includes cytotoxins which disrupt microtubuleassembly and the second class cytotoxins which target DNA structure.Accordingly, cytotoxins which may e.g. be covalently coupled to thelinker as disclosed above include doxorubicin, calicheamicin,auristatin, maytansine duoarmycin and analogs thereof, α-amaitin,tubulysin and analogs thereof. Methods for covalently coupling orattaching cytotoxins to linkers are known in the art and may e.g. bedone according to the method disclosed in Mol. Pharmaceutics 2015, 12,1813-1835.

In one embodiment the at least one linker as disclosed above iscovalently coupled to at least one Fab or scFv light chain (VL) of theinventive heterodimeric bispecific immunoglobulin molecule. Accordinglyat least one light chain, e.g. one or two light chains of the inventiveheterodimeric bispecific immunoglobulin molecule may be coupled to alinker as disclosed above. For example, covalent coupling may be done byintroducing, one or more, e.g. 2, 3, or 4, 5 or 6, additional cysteineresidues into the scFv molecule, mainly at the C-terminus, which allowconjugation to sulfhydryl-reactive reagents as disclosed in e.g. Martyet al. Protein Expression and Purification 21, 156-154 (2001);Nataranja, A et al., Bioconjugate Chem. 16, 113-121; Krimner et al.Protein Eng., Des. Sel. 19, 461-470; Albrecht et al. Bioconjugate Chem.15, 16-26), Cysteine residues can e.g. also be alkylated by reactingthem with α-haloketones or Michael acceptors, such as maleimidederivates. Alternatively, the modification of lysine residues may e.g.be utilized which is the oldest and and most straightforward method forlabeling proteins via the primary lysine amino groups. The ε-amino groupof lysine within the protein of interest can be readily reacted withactivated esters, sulfonyl chlorides, isocyanates and isothiocyanates toresult in the corresponding amides, sulfonamides, ureas and thioureas(see e.g. Takaoka at el., Angew. Chem. Int. Ed. 2013, 52, 4088-4106).Further examples for bioconjugation include the conjugation offluorescent proteins, dyes, or the tethering with functional molecules,e.g. PEGs, porphyrins, peptides, peptide nucleic acids, and drugs(Takaoka et al., Angew. Chem. Int. Ed. 2013, 52, 4088-4106).

For example, enzyme-mediated conjugation may also be applied forcovalently coupling the linker as disclosed above to the inventiveheterodimeric bispecific immunoglobulin molecule. For example, WO2014/001325 A1 discloses the use of sortase A for site-specificbioconjugation to Fc regions of an antibody. Sortase A (SrtA) is abacterial integral membrane protein first described in Staphylococcusaureus. SrtA catalyzes a transpeptidation reaction anchoring proteins tothe bacterial cell wall. Upon recognition of a sorting signal LPXTG,(X=D, E, A, N, Q, or K) a catalytic cysteine cleaves the peptide bondbetween residues T and G which results in the formation of a thioacylintermediate. This thioacyl intermediate subsequently then can reactswith an amino-terminal glycine acting as a nucleophile. SrtA acceptsN-terminal (oligo)glycine as a nucleophiles, creating a new peptide bondbetween two molecules. SrtA functions at physiological conditions andhas been used for bioconjugation reactions to label proteins with e.g.biotin, or to functionalize a HER2-specific recombinant Fab with theplant cytotoxin gelonin (see e.g. Popp et al. (2011) Angew Chemie Int.Ed. 50: 5024-5032 Kornberger et al (2014) mAbs 6 (2): 354-366).Typically, target proteins such as e.g. the VL and VH chains of thefirst and/or second Fab or scFv fragments as disclosed above, arelabeled carboxyterminally with the LPXTG motif followed by apurification tag such that the SrtA-mediated transpeptidation removesthe purification tag and generates the labeled protein.

In one embodiment the heterodimeric bispecific immunoglobulin moleculeaccording to the invention as disclosed above comprises two linkerscovalently coupled to the Fab or scFv light chains of said heterodimericbispecific immunoglobulin molecule. For example, the linkers may becoupled to the light chain of the VL chain of the first Fab or scFvfragment of the inventive the heterodimeric bispecific immunoglobulinmolecule which specifically binds to EGFR as disclosed above and e.g. tothe VL chain of the second Fab or scFv fragment of the inventive theheterodimeric bispecific immunoglobulin molecule which specificallybinds to c-MET as disclosed above.

In one embodiment the Fab or scFv light chains and/or the CH3 domainsand/or the CH2 domains of the heterodimeric bispecific immunoglobulinmolecule of the invention as disclosed above are covalently coupled to alinker, whereby said linker is covalently coupled to a dye,radioisotope, or cytotoxin as disclosed above. For example, the VLchains of the first and second Fab or scFv fragment may be covalentlycoupled to a linker as disclosed above, whereby the linker is furthercoupled to a dye radioisotope or cytotoxin as disclosed above, or bothengineered CH3 domains of the inventive heterodimeric bispecificimmunoglobulin molecule as disclosed above (“AG-SEED”, “GA-SEED”) may becovalently coupled to a linker as disclosed above, or e.g. the CH2domains of the heterodimeric bispecific immunoglobulin molecule of theinvention as disclosed above, may each be covalently coupled to a linkeras disclosed above. Studies with anti-CD30 monoclonalantibody—auristatin E (MMAE) conjugates have shown that ADCs with aantibody:drug stoichiometry of 1:2-1:4 are most effective, with a ratioof 1:4 being most preferable (see e.g. Hamblett et al. Clinical CancerResearch (2004) Vol. 10, 7063-7070). Thus, the inventive heterodimericbispecific immunoglobulin molecule as disclosed above may e.g. comprises2, 3, or 4 linker molecules which are covalently coupled to theinventive heterodimeric bispecific immunoglobulin molecule, whereby eachlinker is preferably coupled to a cytotoxin as disclosed above, e.g. theVL chains and the VH chains of the heterodimeric bispecificimmunoglobulin molecule of the invention may be coupled to a cytotoxinvia a linker as disclosed above. For example, the VH chains and the CH3domains of the inventive heterodimeric bispecific immunoglobulinmolecule as disclosed above may be covalently coupled to a linker,whereby each linker is further coupled to a cytotoxin. Alternatively,the VL chains of the first and second Fab or scFv fragment and the CH3or CH2 domains of the inventive heterodimeric bispecific immunoglobulinmolecule as disclosed above may be covalently coupled to a linker whichis further coupled to a cytotoxin as disclosed above.

In one embodiment the heterodimeric bispecific immunoglobulin moleculeaccording to the invention as disclosed above is for use in thetreatment of cancer. The term “cancer” as used in the present inventionrefers to a variety of conditions caused by the abnormal, uncontrolledgrowth of cells, e.g. cells capable of causing cancer, referred to as“cancer cells”, possess characteristic properties such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and/or certain typical morphological features.Cancer cells may e.g. be in the form of a tumor, but such cells may alsoexist singly within a subject, or may be a non-tumorigenic cancer cell.The term cancer as used in the context of the inventive method oftreatment may e.g. refer to prostate cancer, breast cancer, adrenalcancer, leukemia, lymphoma, myeloma, bone and connective tissue sarcoma,brain tumors, thyroid cancer, pancreatic cancer, pituitary cancer, eyecancer, vaginal cancer, vulvar cancer, cervical cancer, uterine cancer,ovarian cancer, esophageal cancer, stomach cancer, colon cancer, rectalcancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lungcancer, testicular cancer, penal cancer, oral cancer, skin cancer,kidney cancers, Wilms' tumor and bladder cancer, metastatic (mCRC),non-resctable liver metastases, squamous cell carcinoma of the head andneck, non-small cell lung cancer (NSCLC), head and neck squamous cellcarcinoma (HNSCC), Merkel cell carcinoma.

In one embodiment the invention provides a composition which comprisesthe heterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above and at least one further ingredient. For example, theinventive composition may comprise the heterodimeric bispecificimmunoglobulin molecule of the invention as disclosed above and one ormore of water, buffer, stabilizer, salt, sugar, preservative (e.g.benzalkonium chloride), lipids, anti-oxidants, carboxylic acids,polyethylene, glycol (PEG). For example, the buffer or buffer solutionmay have a pH from about 5 to about 9, e.g. from about pH 5 to about pH6, or from about pH 6 to about pH 7, or from about pH 8 to about pH 9,or from about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8 to about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 andmay e.g. comprise sodium acetate, histidine, citrate, succinate orphosphate buffers. For example, sodium acetate, histidine, citrate,succinate or phosphate may be present in the composition according tothe invention in a concentration of from about 10 mM, 15 mM, 20 mM, 25mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM to about 60 mM, 70 mM, 80 mM, 90mM, 100 mM, 125 mM, 150 mM. For example, the buffer solutions asdisclosed above may be combined with a preservative such as benzalkoniumchloride to stabilize the inventive heterodimeric bispecificimmunoglobulin molecule as disclosed above. Other ingredients may e.g.include, polyethylene, glycol with an average molecular weights of200-4000 Dalton, e.g. 300, 400, 500 600, 700, 800, 900, 1000, 1500,1750, 2000, 2250, 2500, 3000, 3500 Dalton and its derivatives.Polyethylene glycol derivatives may e.g. also be used and may e.g.include polyethylene glycol monolaurate, polyethylene glycol mono-oleateand polyethylene glycol monopalmitate. For example, the compositionaccording to the invention may comprise the inventive heterodimericbispecific immunoglobulin molecule as disclosed above in aqueous orlyophilized form and at least one further chemotherapeutic agent,wherein the agent is selected from the group comprising capecitabine,5-fluoro-2′-deoxyuiridine, irinotecan, 6-mercaptopurine (6-MP),cladribine, clofarabine, cytarabine, floxuridine, fludarabine,gemcitabine, hydroxyurea, methotrexate, bleomycin, paclitaxel,chlorambucil, mitoxantrone, camptothecin, topotecan, teniposide,colcemid, colchicine, pemetrexed, pentostatin, thioguanine; leucovorin,cisplatin, carboplatin, oxaliplatin, or a combination of 5-FU,leucovorin, a combination of 5-fluorouracil/folinic acid (5-FU/FA), acombination of 5-fluorouracil/folinic acid (5-FU/FA) and oxaliplatin(FLOX), a combination of 5-FU, leucovorin, oxaliplatin (FOLFOX), or acombination of 5-FU, leucovorin, and irinotecan (FOLFIRI), or acombination of leucovorin, 5-FU oxaliplatin, and irinotecan (FOLFOXIRI),or a combination of Capecitabine and oxaliplatin (CapeOx). in oneembodiment the present invention provides a pharmaceutical compositionwhich comprises the heterodimeric bispecific immunoglobulin molecule ofthe invention as disclosed above and at least one further ingredient, orwhich comprises the inventive composition as disclosed above. Forexample, the pharmaceutical composition of the invention may comprisethe heterodimeric bispecific immunoglobulin molecule of the invention asdisclosed above in a concentration from about 10 mg/ml, 20 mg/ml, 25mg/ml, 30 mg/ml, 35 mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml, 60 mg/ml toabout 70 mg/ml, 75 mg/ml, 80 mg/ml, 100 mg/ml, 112 mg/ml, 125 mg/ml, 150mg/ml, 175 mg/ml, 200 mg/ml, or e.g. from about 10 mg/ml to about 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55mg/ml, 60 mg/ml to about 70 mg/ml, 75 mg/ml, 80 mg/ml, 90 mg/ml, 100mg/ml, 112 mg/ml, 125 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, or e.g. 20mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55mg/ml, 60 mg/ml, to about 70 mg/ml, 75 mg/ml, 80 mg/ml, 90 mg/ml, 100mg/ml, 112 mg/ml, 125 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, and e.g.an aqueous buffer as disclosed above. The inventive pharmaceuticalcomposition as disclosed above, may e.g. also comprise surfactants suche.g. anionic surfactants such as e.g. a mixture of sodium alkylsulfates, cationic surfactants, such as e.g. quaternary ammonium andpyridinium cationic surfactants, or non-ionic surfactants, such as e.g.Sorbitan esters, polysorbates, e.g. Polysorbat 20(Polyoxyethylen-(20)-sorbitanmonolaurat), Polysorbat 21(Polyoxyethylen-(4)-sorbitanmonolaurat), Polysorbat 40(Polyoxyethylen-(20)-sorbitanmonopalmitat), Polysorbat 60(Polyoxyethylen-(20)-sorbitan-monostearat), Polysorbat 61(Polyoxyethylen-(4)-sorbitanmonostearat), Polysorbat 65(Polyoxyethylen-(20)-sorbitantristearat), Polysorbat 80(Polyoxyethylen-(20)-sorbitanmonooleat), Polysorbat 81(Polyoxyethylen-(5)-sorbitanmonooleat) Polysorbat 85(Polyoxyethylen-(20)-sorbitantrioleat), Polysorbat 120(Polyoxyethylen-(20)-sorbitanmonoisostearat), poloxamers e.g. poloxamer105, poloxarner 108, poloxamer 122, poloxamer 124, poloxamer 105benzoate. Perservatives which may be comprised in the pharmaceuticalcomposition according to the invention may be benzalkonium chlorid in aconcentration of 0.004% to 0.01%. For example, the inventivepharmaceutical composition may be formulated by use of conventionaltechniques as discrete dosage forms, such as capsules, a solution or asuspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus;together with suitable pharmaceutically acceptable carrier.

In one embodiment the pharmaceutical composition of the invention asdisclosed above is for use in the treatment of cancer. For example, theinventive pharmaceutical composition as disclosed above for use in thetreatment of cancer may be administered to a person inflicted withcancer.

In one embodiment the invention provides a method of treatment whichcomprises administering to a subject a therapeutically effective amountof the inventive pharmaceutical composition as disclosed above. Forexample, the inventive method of treatment may comprise administering aperson in need thereof afflicted with cancer as disclosed above fromabout 0.001 mg/kg to about 50 mg/kg of the inventive pharmaceuticalcomposition, or from about 0.005 mg/kg to about 45 mg/kg, or from about0.01 mg/kg to about 40 mg/kg, or from about 0.05 mg/kg to about 35mg/kg, or from about 0.1 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg,8 mg/kg, 9 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg,22.5 mg/kg, 25 mg/kg to about 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30mg/kg, 32.5 mg/kg, 35 mg/kg, 37.5 mg/kg, 40 mg/kg, 42.5 mg/kg, 45 mg/kg.As used the term “mg/kg” refers to mg of the inventive pharmaceuticalcomposition/kg body weight in the present invention. For example, apharmaceutically effective amount of the inventive pharmaceuticalcomposition may be administered to an individual inflicted with cancer.The pharmaceutically effective amount depends on the individual, thetype of cancer to be treated, the body weight and age of the individual,the level of the disease or the administration route.

EXAMPLES Example 1: Generation of Anti c-Met and Anti-EGFR Binders

Generation of c-MET Binders

Panning of naïve phage display antibody gene libraries HAL718 againsthuman c-MET was performed according to Hust and colleagues. 36; 37Briefly, after pre-selection with panning buffer (1% skim milk powder,1% BSA, 0.05% Tween®20 in PBS) in maxisorp 96 well plates (Nunc), scFvdisplaying phages were selected on 1 μg immobilized c-MET-Fc (R&DSystems, 358-MT/CF) or c-MET SEMA domain (produced in house) and elutedwith trypsin. After two to three rounds of panning, c-MET specificbinders were enriched and screened by capture c-MET ELISA of producedscFv.

For affinity maturation, (a) error prone PCR for variable domains wasperformed using the GeneMorph II Random Mutagenesis Kits (AgilentTechnologies) according to the manufacturer's instruction, (b)randomization of complementary-determining region three of the heavychain (CDR-H3) ordered by GeneArt applying a parsimonious mutagenesisstrategy 70, and (c) light chain shuffling using the diversity of theHAL7/8 were conducted. Panning was carried out using phage display andyeast display for F06 and B10, respectively. For clone F06, an off-ratescreening strategy was applied by stringent washing (ten times) with 100μl panning buffer per well as well as adding soluble c-MET forcompetition (starting in the second round). CS06 was based on rationalcombination of abundant mutations from approach (a) and (b), B10v5 wasderived from approach (c) using yeast surface display as described ine.g. Biotechnol. Bioeng. 2009 103: 1192-201; Protein Eng Des Sel 2010;23: 155-9.

Generation of Anti-EGFR Binders

The structure of C225 bound to the extracellular domain of EGFR 42 wasoptimized with the Rosetta Protein Structure and Design program (version2.3.0) using a fixed backbone protocol and side chain optimization tominimize the energy of the starting model for design according to theRosetta energy function. Interfacial water molecules observed in thecrystal structure were retained during the minimization, but not duringsubsequent design calculations. Thirty-seven residues at or near theantibody-antigen interface were selected for a saturating, in silicopoint mutagenesis. At each of these residues 19 variants were created(wild type and 18 mutations, no cysteine) optimizing the rotamer of themutated residue while keeping the backbone fixed. Using thesepreliminary models, neighbour residues were identified as any residuewith at least 3 heavy atoms within 5.5 Å of a heavy atom on the designresidue. The rotamer of the mutated residue and its neighbours wereoptimized using the standard Rosetta score function (a linearcombination of terms including a Lennard-Jones potential, an orientationdependent hydrogen bonding potential an implicit solvation model andstatistical terms that capture backbone-dependent amino acid and rotamerpreferences. The hydrophobic substitutions will be described elsewhere.The polar substitutions were filtered to only those variants withimprovements of at least 0.5 Rosetta energy units in either theorientation-dependent hydrogen bonding score or the pair potentialrelative to the repacked native to select improved variants. The threeaffinity enhancing point substitutions were combined into a triplemutant, and this was repacked and scored by Rosetta as described abovefor the point mutants. The affinity of the selected variants wasmeasured in vitro by surface plasmon resonance. The variants were alsotransferred to the hu225 scFv and the affinities in this context wereverified by biolayer interferometry.

Example 2: Expression and Purification of Bispecific c-MET×EGFRSEEDbodies

Several combinations of EGFR and c-MET antibody fragments according tothe invention as disclosed herein were joined to bispecific antibodiesusing the SEED-technology. Bispecific c-MET×EGFR SEEDbodies wereexpressed by transient transfection of Expi293F™ cells (human embryonickidney cells) according to the manufacturer's instruction of thetransfection kit (Invitrogen). Briefly, suspension Expi293F™ cells werecultured in Expi293F™ expression medium (Invitrogen) at 37° C., 5% CO₂and 180 rpm. On the day of the transfection, cells were seeded in freshmedium with a density of final 2×10⁶ viable cells/ml.DNA-ExpiFectamine™293 reagent mixture diluted in Opti-MEM® I medium(Invitrogen) was added to the cells. 16 h post transfection,ExpiFectamine™293 transfection enhancer 1 and 2 were added. Cellsupernatants containing secreted antibodies were harvested 5 days aftertransfection by centrifugation at 4,300×g, 4° C. and 20 min andfiltration through 0.22 μm Stericup or Steriflip devices (Millipore).Small scale productions were performed in a volume of 25 ml andpurification was carried out with PROSEP® A centrifugal Protein Acolumns (Millipore, #P36486) according to manufacturers' instructionsfollowed by dialysis to PBS pH 7.4 using Pur-A-Lyzer™ Dialysis Kit(Sigma-Aldrich).

Large scale productions were performed in an expression volume of 200ml. Supernatants were purified by affinity chromatography (5 ml HiTrapMabSelect SuRe, GE Healthcare) on an AKTA Explorer 100 (GE Healthcare)with subsequent preparative size exclusion chromatography (HiLoad26/60Superdex 200 pg, GE Healthcare). Protein concentrations weredetermined by UV A280 spectroscopy and purity was analyzed by gelelectrophoresis with 4%/8% NuPAGE BisTris gels (Life technologies) andcoomassie staining as well as analytical size exclusion high performanceliquid chromatography (TSK Super SW3000, Tosoh). Endotoxin levels wereassessed by Limulus amebocyte lysate Endosafe® PTS cartridges andEndosafe® PTS reader (Charles River).

Antibody VH and sequences for humanized oa 5D5 (MetMAb, onartuzumab),LY2875358 (LA480_vC8H241, emibetuzumab), and h224G11 (ABT-700) werederived from publicly available information (e.g. U.S. Pat. Nos.6,214,344B1, 8,398,974 B2, 0,273,060A1). Sequences were cloned inmammalian expression vectors containing constant IgG1 light and heavychain fragments except in case of oa 5D5 knob-into-hole technology wasapplied (e.g. as disclosed in Protein Eng 1996; 9: 617-21). Allanti-c-MET reference antibodies as well as cetuximab (C225, Erbitux) andmatuzumab were produced in-house (Merck) in HEK293E cells using standardtransfection and purification procedure e.g. as described above.

Example 3 : Binding of Bispecific c-MET×EGFR Antibodies to c-MET andEGER on Cells

Bispecific c-MET×EGFR antibodies, one-armed (monovalent) controlantibodies (anti-c-MET and anti-EGFR) as well as a non-related isotypecontrol (anti-hen egg lysozyme, anti-HEL) were tested for their bindingto c-MET and EGFR expressing NCI-H441 cells (as e.g. shown in FIG. 1,FIG. 15). NCI-H441 cells were detached with trypsin, centrifuged at250×g for 10 min at 4° C. and resuspended in FACS buffer (1% BSA 1×PBS).Cells were transferred to in 96 well round bottom plates at a density of1×10⁵ cells/well on ice. Purified c-MET×EGFR bispecific antibodies(0.02-200 mM) were added in FACS buffer in triplicates for 1 h on ice.Cells were centrifuged for 1000×g for 5 min at 4° C. and washed 3 timeswith 100 μl FACS buffer. Cells were incubated with 500 ng/wellFluorescein (FITC)-conjugated goat anti-human Fc gamma fragment IgGspecific antibody (Jackson ImmunoResearch) diluted in FACS buffer on icefor 1 h. Cells were washed again 3 times with 100 μl FACS buffer. Forcounter staining of non-viable cells, centrifuged were resuspended in200 μl propidium iodide solution (Invitrogen) diluted in FACS buffer(1:200). Cell were analyzed for fluorescence at 488 nm using a GuavaeasyCyte HT cytometer (Millipore). Data were plotted as meanfluorescence intensity (raw fluorescence substracted by background, e.g.non-stained cell control) against the logarithm of the bispecificantibody concentration and fitted to a sigmoidal dose-response curvewith variable slope using GraphPad Prism 4 (GraphPad Software).

Example 4: Epitope Binning of c-MET Binders Using Bio-LayerInterferometry (BLI)

An epitope binning experiment was carried out with c-MET antibodieswhich were used in the bispecific antibodies and compared to referenceantibodies from the literature (MetMAb, Emibetuzumab, h224G11).Biosensor experiments using bio-layer interferometry were performed onan Octet Red platform (Forté Bio) equipped with anti-human Fc(AHC)biosensor tips (Forté Bio). All data were collected at 30° C. inkinetics buffer (PBS pH 7.4, 0.1% BSA, 0.02% Tween-20. Human c-METECD-His (HGFR, hepatocyte growth factor receptor extracellular domain)was produced and purified in-house. Biosensor tips were equilibrated 30sec in PBS. Then, 25 nM for bivalent IgGs and 50 nM for monovalentone-armed antibodies in PBS were immobilized on biosensor tips for 200sec as primary antibody. Tips were quenched with 400 nM of a non-relatedcontrol antibody (anti-hen egg lysozyme, anti-HEL SEED, diluted in PBS)to minimize subsequent binding of secondary antibodies to biosensortips. Following acquisition of a baseline in kinetics buffer for 60 sec,human c-MET-ECD was subjected to immobilized primary antibodies for 600sec. Afterwards, interactions of secondary anti-c-MET antibodies toc-MET-ECD bound to immobilized primary antibodies was analyzed for 600sec Analysis of secondary antibody binding was analyzed visually bydistinguishing simultaneous binding characterized by a higher bindingrate [nM] compared to a non-related isotype control (anti-HEL SEED). Theresults of the epitope binning are depicted in FIG. 2A.

Example 5: HGF Competition ELISA Assay/HGF Displacement by MonoclonalAntibodies

Competition of recombinant human HGF (Hepatocyte growth factor, R&DSystems, 294-HGN/CF) with antibody binding to recombinant human c-METECD (HGFR extracellular domain, Hepatocyte growth factor receptor) wasdetected by ELISA using HGF in solid phase, Recombinant human HGF (1.255pmol) was immobilized on 96 well Maxisorp plates (Thermo Scientific)overnight at 4° C. After blocking plates with 2% BSA, biotinylatedrecombinant human c-MET ECD (1.13 pmol) pre-incubated with serialdilutions of antibodies (200 nM to 0.2 nM) were added to plates. Bindingwas revealed using HRP-conjugated strepatvidin (Merck Millipore) and TMBsubstrate and sulfuric acid (1 step UltraTMB ELISA solution). Resultingabsorbance for c-MET ECD binding to HGF without addition of anti-c-METdirected antibody was defined as 100% HGF binding. Anti-HEL (hen egglysozyme) was used as an unrelated isotype control antibody. Data wereplotted as % HGF binding against the logarithm of the antibodyconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software). The resultsof the displacement are depicted in FIG. 3.

Example 6: Cell Titer Glow Assay

Cell viability was quantified using the cell titer glow assay (Promega)and was performed according to the manufacturer's instructions. Briefly,cells were detached and seeded in the inner wells of opaque white tissueculture treated 96 well plates (Perkin&Elmer). The seeding cell numberranged from 8,000 to 15,000 viable cells per well depending on the cellline in 80 μl per well. Cells were allowed to attach at least threehours in a humidified chamber at 37° C., 5% CO2. Then, cells weretreated with antibodies in duplicates which were diluted in cell linespecific medium (ranging from 60 to 0.01 nM final). Depending on theassay, Fab-toxin conjugates were added in a threefold molar excess(Fab-toxin from Moradec, MMAE or DMSA). After 72 hours, viability ofcells was detected by adding 100 μl per well of CellTiter-Glo® reagent(Promega) with subsequent mixing on a plate shaker for two minutes at350 rpm and 10 min incubation in the dark at room temperature.Luminescence was measured at a Synergy 5 (Biotek) with a read time of0.5 seconds per well (sensitivity: 170). Background luminescence inwells with only medium with the CellTiter-Glo® reagent (Promega) wassubtracted. Data were plotted as percentage of untreated cell viabilityagainst the logarithm of antibody concentration and fitted to asigmoidal dose-response curve with variable slope using Graph pad Prism4 (GraphPad Software).

Example 7: ADC Generation and Antibody Dependent Cellular CytotoxicityADC Generation

Sortase mediated site-directed conjugation of valine-citrulline(vc)-monomethyl auristatin E (MMAE) to antibody Fc was performed asdescribed elsewhere (see e.g. ACS Chem.Biol. 2015; 10: 2158-65).Briefly, antibodies or the inventive heterodimeric bispecificimmunoglobulin molecules carrying enzyme recognition site C-terminallyon both heavy chains were generated, transfected and purified byaffinity chromatography. Then, one equivalent of antibody was incubatedwith 11 equivalents of substrate-vc-MMAE conjugate in the presence of 5μM Sortase and 5 mM CaCl2 in reaction buffer (50 mM Tris, 150 mM NaCl,pH 7.5) for 30 min at 22° C. The reaction was stopped with 10 mM EDTA ascalcium ion chelator. The resulting ADC was purified by size exclusionchromatography.

Antibody Dependent Cellular Cytotoxicity.

Capability of the antibodies to induce ADCC was assessed using fire ADCCReporter Bioassay Core Kit (Promega) according to the manufacturer'sinstruction. Briefly, target cells (A431 cells) were detached and seededinto the inner wells of opaque white tissue culture treated 96 wellplates (Perkin&Elmer) with a cell density of 12,500 viable cells perwell (100 μl), A431 cells were cultured in ADCC buffer containing RPMI1640 medium (Gibco) supplement with 4% low IgG fetal bovine serum (FBS,Gibco). Cells were allowed to attach overnight in a humidified chamberat 37° C., 5% CO2. The next day, medium was removed and cells weretreated with 25 μl antibodies per well diluted in ADCC buffer (finalconcentrations ranging from 5 to 0.0016 nM). Afterwards, recombinantJurkat cells (Promega) were added which function as effector cells (360μl effector cells diluted in 3.6 ml ADCC buffer, 25 μl per well). Aftersix hours of incubation in a humidified chamber at 37° C., CO2, 75 μl ofBio Glo Luciferase Substrate (Promega), which was equilibrated at roomtemperature, was added per well. After ten minutes of incubation at roomtemperature protected from light, luminescence was measured at a Synergy5 (Biotek) with a read time of 0.5 seconds per well (sensitivity: 170),Background luminescence in wells with only medium was subtracted.Relative luminescence units were plotted against the logarithm ofantibody concentration and fitted to a sigmoidal dose-response curvewith variable slope using GraphPad Prism 4 (GraphPad Software).

Example 8: Receptor Phosphorylation Assay

To assess the effect of binding of the inventive heterodimericbispecific immunoglobulin molecules on c-MET and EGFR-mediated signalingphosphorylation levels of both c-MET and EGFR were determined by c-METor EGFR capture electrochemiluminescence (ECL) ELISA (MSD assay). Allreagents were obtained from Meso Scale Discovery and prepared accordingto the manufacturer's instructions. Briefly, cells were plated in96-well tissue culture plates (Sigma-Aldrich) one day before treatment,serum starved and treated with serially diluted antibodies (0-167 nM instarvation medium) for 1 h at 37° C., 5% CO2. Upon stimulation witheither 100 ng/ml HGF and/or EGF (both R&D Systems) for 5 min at 37° C.,cells were lysed with ice-cold lysis buffer supplemented with proteaseand phosphatase inhibitors (Calbiochem). High bind 96-well platesincluding electrodes (Meso Scale Discovery) were coated with captureanti-total c-MET (Cell Signaling Technologies) or anti-total EGFRantibodies (Abcam) followed by blocking with 3% Block A in PBSsupplemented with 0.05% Tween®20. After incubation with cell lysates,detection was carried out with anti-phospho c-MET (Cell SignalingTechnologies), anti-phospho-tyrosine antibodies (R&D Systems) and by thesupplier recommended detection substances. Measurements were performedwith the SECTOR® Imager 6000 (Meso Scale Discovery). For quantificationof phospho-AKT levels, the Phospho(Ser473)/Total AKT Assay Whole CellLyate Kit (Meso Scale Discovery) was used. Dose response curves wereplotted as the logarithm of antibody concentration versus ECL signal.IC₅₀ values were calculated by a 3PL fitting model using GraphPad Prism5 (GraphPad Software, Inc.). Data from at least two experiments wereused to calculate mean IC₅₀±standard deviation (s.d.), see e.g. FIG. 20,FIG. 25 (A).

Example 9: Quantification of Cell Surface Receptor Density

Receptor surface expression levels on selected cell lines weredetermined using the QFIKIT (Dako K0078) employing flow cytometry, theresults of which are shown in FIG. 18. Briefly, five populations ofcalibration beads presenting different numbers et mouse mAb molecules ontheir surfaces were used as a calibration standard, 1.5×105 cells/wellwere labeled with primary mouse anti-EGFR (ab187287, Abcam) and mouseanti-c-MET antibodies (MAB3582, R&D Systems) at saturating doses (5μg/ml). Then, beads and cells were stained with secondary goatanti-mouse Fc F(ab′)₂ FITC conjugate (10 μg/ml, Jackson Immune Research)and were subjected to flow cytometry measurement using a Guava easyCyteHT cytometer (Millipore). Beads and cells were measured on the same dayusing the same settings. Based on a calibration line for fluorescence ofbeads versus bead surface density, antigen cell surface densities forc-MET and EGFR were calculated.

Example 10: Internalization Assay

Internalization of the inventive heterodimeric bispecific immunoglobulinmolecules was either determined by flow cytometry using an anti-AlexaFluor 488 quenching antibody or by confocal microscopy applying pHstripping. For flow cytometry, cells (1×105) were incubated with 100 nMbsAbs followed by Alexa Fluor 488 conjugated anti human Fc (Fcγspecific, Jackson Immuno Research). After washing with FACS buffer,cells were incubated at either 37° C. or 4° C. for 1 h allowinginternalization. Afterwards, residual surface binding of bsAb wasquenched by anti-Alexa Fluor 488 IgG (Life Technologies) and cells werefixated with 4% (w/v) formaldehyde (Calbiochem) and subjected to flowcytometric analysis. Internalization was calculated as following:

${{rel}.\mspace{11mu}{{interalization}\mspace{14mu}\lbrack\%\rbrack}} = \frac{\left( {37{^\circ}\mspace{14mu}{C.\mspace{14mu}{with}}\mspace{14mu}{quench}} \right) - \left( {4{^\circ}\mspace{14mu}{C.\mspace{14mu}{with}}\mspace{14mu}{quench}} \right)}{\left( {37{^\circ}\mspace{14mu}{C.\mspace{14mu}{without}}\mspace{14mu}{quench}} \right) \times 100}$

For fluorescence microscopy, cells (3×10⁵) were grown on glasscoverslips (Menzel Glaser) placed in 6 well plates. Two days later,cells were kept on ice and treated with 100 nM bsAbs followed bydetection with Alexa Fluor 488 conjugated anti human Fc Fab fragment.After washing with 1% BSA in PBS, cells were incubated in respectivemedium at either 37° C. or 4° C. for 1 h allowing internalization. Byaddition of ice-cold low pH buffer (50 mM glycine, 150 mM NaCl, pH 2.7adjusted with HCl), residual bsAbs on the cell surface were removed.Finally, cells were fixated with 4% (w/v) formaldehyde and mounted onobject slides with ProLong Diamond Antifade Mountant with DAPI (LifeTechnologies). Analysis was carried out with a Leica TCS SPS confocalmicroscope equipped with a 100×objective (Leica Microsystems).

Example 11: Cell Culture

Human cancer cell lines which were used according to the presentinvention were obtained from the American Type Culture Collection (A431,A549, MDA-MB-468, NCI-H1975, NCI-H441, NCI-H596), the Riken BiorescourseCenter Cell Bank (EBC-1, KP-4), Lipha (HepG2), and German Collection ofMicroorganims and Cell Cultures (MKN45) and maintained according tostandard culture conditions (37° C., 5% CO₂, 95% humidity) usingrecommended media formulations, A549 and A431 were cultivated inDulbecco's Modified Eagle's Medium (DMEM, Life Technologies) containing10% Fetal Bovine Serum (FBS, Life Technologies). MDA-MB-468, NCI-H1975,HepG2, and MKN45 were maintained in RPMI-1640 (Life Technologies)supplemented with 10% FBS, 2 mM L-glutamine and 1 mM sodium pyruvate(both Life Technologies). NCI-H441, NCI-H596 were cultivated inRPMI-1640 with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 2.5 g/LD(+)-glucose (Sigma-Aldrich) and 10 mM HEPES (Life Technologies). KP-4cells were cultivated in DMEM/F-12 with 10% FBS. EBC-1 cells weremaintained in Minimal Essential Medium (MEM) with 10% FBS and 2 mML-glutamine. NHEK.f.-c, (PromoCell, #C-12007) were obtained fromPromoCell and propagated in recommended keratinocyte growth medium withsupplements (PromoCell, #C-20111) and with the DetachKit (PromoCell,#C-41210) for cell detachment. Expi293F™ cells were purchased from LifeTechnologies and cultivated in corresponding Expi293 expression medium.All cell lines were shown to be sterile, certified mycoplasma-free, andnever exceeded passage 20.

Example 12 Surface Plasmon Resonance

Affinity and kinetic parameters of in silico designed C225 variants wasverified by surface plasmon resonance. Computationally guidedsubstitutions were introduced into the wild-type C225 using theQuikChangeII kit (Stratagene) with mutagenic primers. The variantantibodies were expressed in HEK-293-6E cells. Cleared supernatant waspurified by affinity chromatography using protein A. The antibodyconcentration was determined by absorbance at 280 nm, and the purity wasverified by SDS-PAGE analysis. Surface plasmon resonance was performedon a Biacore A-100 (GE Healthcare). CM5 chips were coupled with goatanti-human IgG antibody (Jackson ImmunoResearch, Inc., 109-005-098) andused to capture the wild-type C225 or designed variants. Human EGFR(extracellular domain, R&D Systems, 1095-ER) was used as analyte. Theaffinity was determined by titrating the analyte from 0 to 40 nM anddetermining kinetic rate constants using the BiaEvaluation software tofit the association and dissociation phases using a 1:1 Langmuir bindingmodel. The KD was determined as the ratio of the kinetic constants.

Example 13: Thermal Shift Assay

Thermal stability of the inventive heterodimeric bispecificimmunoglobulin molecules, as well as of controls (C225 (cetuximab),matuzumab and “one-armed” (oa) constructs) was measured using aStepOnePlus Real-Time PCR System (Life Technologies) according to themanufacturer's instructions, the results of which are shown in FIG. 17and the corresponding description. Briefly, 1 μM protein was mixed witha 20 fold excess of SYPRO Orange (Life Technologies) in PBS pH 7.4.Melting curves were recorded from 25° C. to 99° C. with an increment of1° C./60 s. Data were analyzed with the Protein Thermal Shift™ Software(Life technologies) by calculating the maximum of the second derivativecurve.

1-34. (canceled)
 35. An antibody or antigen-binding fragment thereofthat binds to c-MET, comprising: a) a light chain variable region, VL,selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 47, andSEQ ID NO: 51, and a heavy chain variable region, VH, selected from thegroup consisting of SEQ ID NO: 32 and SEQ ID NO: 48; b) a VL of SEQ IDNO: 33, and a VH selected from the group consisting of SEQ ID NO: 34 andSEQ ID NO: 52; or c) a humanized version of the antibody orantigen-binding fragment thereof of a) or b).
 36. The antibody orantigen-binding fragment thereof according to claim 35, wherein saidantibody or antigen-binding fragment thereof is an immunoglobulinmolecule comprising a Fab or scFv fragment that binds to c-MET.
 37. Theantibody or antigen-binding fragment thereof according to claim 35,wherein said antibody or antigen-binding fragment thereof binds c-METwith a K_(D) of at least 5×10⁻⁸ M.
 38. The antibody or antigen-bindingfragment thereof according to claim 35, wherein said antibody orantigen-binding fragment thereof is a one-armed monovalent antibody. 39.The antibody or antigen-binding fragment thereof according to claim 35,comprising: the antibody or antigen-binding fragment of a) or thehumanized version of the antibody or antigen-binding fragment thereof ofa).
 40. The antibody or antigen-binding fragment thereof according toclaim 35, comprising: the VL of SEQ ID NO: 31 and the VH of SEQ ID NO:32; or a humanized version of the antibody or antigen-binding fragmentthereof comprising the VL of SEQ ID NO: 31 and the VH of SEQ ID NO: 32.41. The antibody or antigen-binding fragment thereof according to claim35, comprising: the VL of SEQ ID NO: 51 and the of SEQ ID NO: 32; or ahumanized version of the antibody or antigen-binding fragment thereofcomprising the VL of SEQ ID NO: 51 and the VH of SEQ ID NO:
 32. 42. Theantibody or antigen-binding fragment thereof according to claim 35,comprising: the VL of SEQ ID NO: 47 and the VH of SEQ ID NO: 48; or ahumanized version of the antibody or antigen-binding fragment thereofcomprising the VL of SEQ ID NO: 47 and the VH of SEQ ID NO:
 48. 43. Theantibody or antigen-binding fragment thereof according to claim 35,comprising: the VL of SEQ ID NO: 33 and the VH selected from the groupconsisting of SEQ ID NO: 34 and SEQ ID NO: 52; or a humanized version ofthe antibody or antigen-binding fragment thereof comprising the VL ofSEQ ID NO: 33 and the VH of SEQ ID NO: 34 or
 52. 44. The antibody orantigen-binding fragment thereof according to claim 35, comprising: theVL of SEQ ID NO: 33 and the VH of SEQ ID NO: 34; or a humanized versionof the antibody or antigen-binding fragment comprising the VL of SEQ IDNO: 33 and the VH of SEQ ID NO:
 34. 45. The antibody or antigen-bindingfragment thereof according to claim 35, comprising: the VL of SEQ ID NO:33 and the of SEQ ID NO: 52; or a humanized version of the antibody orantigen-binding fragment thereof comprising the VL of SEQ ID NO: 33 andthe VH of SEQ ID NO: 52.